Alteration in central vascular and cognitive functions during simulated work conditions in males – a secondary analysis from a randomised controlled trial

Poovitha Shruthi Paramshiva; Obhuli Chandran M; Baskaran Chandrasekaran; Vaishali K.; Suresh Sukumar; Rajagopal Kadavigere

doi:10.12688/f1000research.110911.4

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Research Article

Revised

Alteration in central vascular and cognitive functions during simulated work conditions in males – a secondary analysis from a randomised controlled trial

[version 4; peer review: 1 approved, 3 not approved]

Poovitha Shruthi Paramshiva¹, Obhuli Chandran M², Baskaran Chandrasekaran³, Vaishali K.⁴, Suresh Sukumar², Rajagopal Kadavigere ⁵

Poovitha Shruthi Paramshiva¹, Obhuli Chandran M², [...] Baskaran Chandrasekaran³, Vaishali K.⁴, Suresh Sukumar², Rajagopal Kadavigere ⁵

PUBLISHED 06 Apr 2026

Author details Author details

¹ Division of Yoga, Center for Integrative Medicine and Research Manipal Academy of Higher Education, Manipal, karnataka, 576104, India
² Department of Medical Imaging Technology, Manipal College of Health Professions Manipal Academy of Higher Education, manipal, karnataka, 576104, India
³ Department of Exercise and Sports Sciences, Manipal College of Health Professions Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
⁴ Department of Physiotherapy, Manipal College of Health Professions Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
⁵ Department Radio diagnosis and Imaging, Kasturba Medical College Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India

Poovitha Shruthi Paramshiva
Roles: Investigation, Methodology

Obhuli Chandran M
Roles: Conceptualization

Baskaran Chandrasekaran
Roles: Data Curation, Writing – Original Draft Preparation

Vaishali K.
Roles: Conceptualization, Methodology

Suresh Sukumar
Roles: Investigation, Writing – Review & Editing

Rajagopal Kadavigere
Roles: Supervision

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Manipal Academy of Higher Education gateway.

Abstract

Background

Prolonged uninterrupted sitting impairs central vascular hemodynamics, which may compromise cerebral perfusion and cognitive performance. Activity micro-breaks have been proposed to mitigate these effects, yet the association between sitting-induced changes in carotid artery hemodynamics and executive function remains unexplored.

Methods

This secondary analysis of a randomised crossover trial enrolled 17 physically inactive young adult males (age 25–35 years). Participants completed three four-hour simulated work conditions (minimum six-day washout): uninterrupted sitting (SIT); sitting interrupted by three minutes of light-intensity walking hourly (LIT); and sitting interrupted by three minutes of moderate-intensity stair climbing hourly (MIT). Carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — were assessed at baseline, hour 2, and hour 4 via Duplex ultrasound. Executive function was assessed at baseline and hours 1–4 using the Eriksen Flanker task, with reaction time and response accuracy as outcomes. Pearson correlations and multiple linear regression examined associations between changes in carotid hemodynamics and executive function from baseline to hour 4 within each condition.

Results

Uninterrupted sitting produced a significant reduction in carotid artery diameter (−0.03 cm, 95% CI: −0.05, −0.01) and a significant decline in response accuracy (−12.7%, 95% CI: −24.81, −0.66). Moderate-intensity micro-breaks were associated with a significant reduction in carotid shear rate and slower reaction times, while response accuracy was preserved. No significant changes were observed during LIT. No significant association was found between changes in carotid hemodynamics and executive function within any condition.

Conclusion

Uninterrupted sitting impaired carotid artery hemodynamics and response accuracy over four hours, while moderate-intensity micro-breaks preserved accuracy and attenuated shear rate reductions. Carotid hemodynamic changes did not independently predict executive function changes, suggesting additional mechanisms warrant investigation. Findings are preliminary, based on a small male-only sample, and require replication in larger mixed-sex cohorts incorporating direct measures of endothelial vasodilatory capacity.

Keywords

Prolonged sitting, Central vascular function, Blood flow, Velocity, Doppler, Micro-breaks, Cognition

Corresponding author: Rajagopal Kadavigere

Competing interests: No competing interests were disclosed.

Grant information: Baskaran Chandrasekaran received a faculty seed grant from Manipal Academy of Higher Education for the present research.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2026 Shruthi Paramshiva P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Shruthi Paramshiva P, Chandran M O, Chandrasekaran B et al. Alteration in central vascular and cognitive functions during simulated work conditions in males – a secondary analysis from a randomised controlled trial [version 4; peer review: 1 approved, 3 not approved]. F1000Research 2026, 11:397 (https://doi.org/10.12688/f1000research.110911.4) First published: 06 Apr 2022, 11:397 (https://doi.org/10.12688/f1000research.110911.1) Latest published: 06 Apr 2026, 11:397 (https://doi.org/10.12688/f1000research.110911.4)

Revised Amendments from Version 3

This manuscript has undergone substantial revision in response to peer review. Key changes include: operational definition of central vascular function as carotid artery diameter, blood flow velocity, and shear rate throughout; removal of overclaims regarding endothelial function, which was not directly measured; replacement of review papers with primary sources in the Introduction; clarification that femoral artery data fall outside the scope of this secondary analysis; replacement of paired t-tests with repeated-measures ANOVA with Condition × Time interactions as the primary statistical test; correction of a systematic change score direction error (baseline minus hour 4 recalculated as hour 4 minus baseline) that had caused the abstract and conclusion to misrepresent the direction of key findings; restructuring of the Results section variable by variable for clarity; and comprehensive revision of the abstract and conclusion to accurately reflect the study design, sample characteristics, and findings. The male-only sample is acknowledged as a significant limitation arising from inadequate culturally adapted recruitment procedures, and the absence of a direct endothelial function measure is noted as a priority for future work.

See the authors' detailed response to the review by Kevin S Heffernan

1. Introduction

Profound dynamic changes in the endothelial functions in early adulthood is associated with early atherosclerosis and cardiovascular disease risk in late adulthood.¹^–³ High sedentary behavior (sitting activities with low energy expenditure less than 1.5 metabolic equivalents) is now identified to be the independent risk factor for the early atherogenesis and cardio-metabolic disease risk.⁴^,⁵ However, high sedentary behavior is evident and continue to rise globally and in contextual settings such as workplace and schools.⁶^–⁸ Hence it is imperative to address contextual sedentary behavior through administering movement based micro-breaks.⁹^,¹⁰ Empirical evidence elucidating the efficacy of advocating activity breaks during prolonged sitting on cardiometabolic disease risk and cognitive decline remains mixed; while some studies report improvements in vascular function and cognitive performance following activity micro-breaks, others report negligible or no significant effects, with discrepancies attributed to variability in break frequency, intensity, and the populations studied.¹¹^–¹³

Central vascular function is operationally defined here as the structural and functional properties of large central conduit arteries, including endothelial vasodilatory capacity (assessed via flow-mediated dilation), arterial wall stiffness (assessed via pulse wave velocity), and carotid artery hemodynamics (diameter, blood flow velocity, and shear rate) — plays a key role in regulating downstream cerebral perfusion and, consequently, cognitive performance.¹⁴^,¹⁵ Primary studies have demonstrated that acute bouts of prolonged sitting progressively impair peripheral endothelial function, evidenced by significant reductions in femoral artery flow-mediated dilation and shear rate.^16,17 Prolonged sitting has additionally been shown to increase central arterial stiffness, with carotid-to-femoral pulse wave velocity rising significantly after sustained sitting periods.¹⁵ With respect to cerebral hemodynamics specifically, Carter et al. demonstrated that prolonged sitting significantly reduced middle cerebral artery blood flow velocity, an effect attenuated by regular walking micro-breaks.⁹ Elevated carotid arterial stiffness has further been linked to reduced cerebral oxygenation during executive function tasks and impaired executive function performance in observational studies.^18,19 While several primary studies have examined the acute effects of prolonged sitting and exercise snacks on endothelial function, arterial stiffness, and cerebral blood flow separately, the integrated relationship between sitting-induced changes in carotid hemodynamics and executive function performance has yet to be directly examined.¹⁹ Executive functions and working memory underpin key work-related skills including decision-making and problem-solving, with potential implications for occupational productivity.^20,21

The present study aimed to examine whether acute changes in central vascular function — specifically, sitting-induced alterations in carotid artery diameter, blood flow velocity, and shear rate — were associated with concurrent changes in executive function performance across uninterrupted sitting and activity micro-break conditions. We hypothesised that within each work condition, greater reductions in carotid artery hemodynamics (diameter, velocity, and shear rate) would be associated with greater deterioration in executive function performance (slower reaction times and reduced accuracy), and that this association would be most pronounced in the uninterrupted sitting condition (SIT) relative to the light- (LIT) and moderate-intensity (MIT) micro-break conditions.

2. Methods

The present findings are the results of the secondary analysis of our ongoing randomised controlled trial. Kasturba Hospitals Institutional Ethics Committee (IEC 383-2021) approved the study and was prospectively registered in India’s Clinical Registry (CTRI/2021/09/036496). The study was conducted and conforms to the research principles of the Declaration of Helsinki.²² All participants provided written informed consent to participate in this study.

2.1 Study design

The findings of the study are derived from a randomized crossover trial that investigates the effects of passive and active work conditions on central vascular functions and cognitive control in young adults.

The study was conducted in the ultrasound testing room, department of radiodiagnosis and imaging of a multidisciplinary teaching hospital. The study was conducted between September 2021 – December 2021. Potential participants were randomised to one sedentary and two active work conditions for four hours, with each study visit was separated by a wash-out period of at least six days. The whole study design is depicted in Figure 1. The present study explored the relation between the dynamic carotid vascular changes (diameter, velocity, shear rate and blood flow) and executive functions.

Figure 1. Design of the present study.

The participants were randomised to the three different behavioral interventions in simulated work conditions for four hours: (1) SIT (uninterrupted sitting for four hours with bathroom breaks only); (2) LIT (sitting interrupted with light intensity walking breaks for three minutes every one hour for four hours of the study period; MIT (sitting interrupted moderate-intensity stair climbing breaks for three minutes every one hour for four hours of the study period). Doppler ultrasound-mediated carotid, superficial femoral artery diameter and velocity were measured every two hours of each visit period, and computer-based executive functions were measured every hour of each visit period.

2.2 Participants

Young adults of age 25 – 35 years of both genders belonging to a single university were invited to the study through the official Exchange student emails, brochures in college and hostel noticeboards. Although participants of all genders were invited, only male students volunteered. The study was conducted in a clinical ultrasound setting requiring exposure of the lower limb for femoral artery Doppler imaging, and female students did not volunteer despite open recruitment. We acknowledge that the absence of a culturally adapted protocol — such as female sonographers, private examination rooms, or gender-specific recruitment strategies — likely contributed to this participation gap and represents a significant limitation of the study design. To be eligible, the potential participants should be physically inactive (self-reported physical activity levels of less than 600 METmin/week International Physical Activity Questionnaire IPAQ), optimum levels of visual acuity (> 5/6) for operating the computer-based cognitive tests with adequate lower limb strength to walk or climb stairs for three minutes. Participants with self-reported cardiometabolic diseases, recent musculoskeletal trauma < three weeks or self-reported depressive disorders affecting physical activity or cognitive measurement were excluded from the study. Further, the volunteers willing to dedicate at least six weekday hours were included in the study.

2.3 Measures

2.3.1 Vascular functions

The vascular functions using Doppler ultrasound was measured as stated and administered in the earlier studies.⁹^,¹⁶^,¹⁷^,¹⁹^,²³^–²⁵ Dynamic carotid artery hemodynamics (right common carotid artery diameter, blood flow velocity, and shear rate) were measured using Duplex ultrasound (GE Voluson Swift, Germany) at a pulsed frequency of 50 MHz with the probe insonated at an angle of 60°. The diameter and velocity were automatically measured using inbuilt edge-detection software, and captured images were analysed using B-mode imaging. Shear rate was calculated using the formula: 4 × [mean blood velocity/arterial diameter]. Maximal blood flow was calculated from continuous diameter and mean blood velocity recordings using: 3.14 × (diameter/2)² × mean blood velocity × 60. Although superficial femoral artery hemodynamics were also recorded as part of the parent randomised controlled trial — where peripheral lower-limb vascular function was a co-primary outcome — femoral artery data are not reported in the present secondary analysis, which is focused specifically on the association between central (carotid) vascular hemodynamics and executive function.²⁶

2.3.2 Executive functions

Executive functions or cognitive control was assessed using the computer-based Response Inhibition test, Eriksen Flanker paradigm (Milliseconds, Inquist). The participants were presented with the visual stimuli on a laptop using Inquist 6.0 software (Millisecond Software, Seattle, WA, USA).

The participants were shown a line with five alphabets H, K, S & C with the central alphabet as the target and flankers on the sides. The participants were instructed to focus on the central alphabet and press either Q or P based on the H or K, and S or C displayed on the left and right sides of the computer screen, respectively. The task had two types of trials: congruent (noise) and incongruent (no noise) trials. All the alphabets are the same in congruent trials, while the flanker and target alphabets were different in the incongruent trials. A total of 120 trials (60 congruent, 60 incongruent) were presented to the participants, which were randomly sampled. Each trial lasted for 3000 ms, with an interstimulus interval of 1500 ms. The participants were given a one min break after every 40 trials. The data were automatically stored in the inquisit software and retrieved as csv files later for analysis.

2.3.3 Anthropometric measures

Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer with participants standing barefoot and positioned in the Frankfort horizontal plane. Body mass was measured to the nearest 0.1 kg using a calibrated digital weighing scale with participants in light clothing and bladder voided. Body mass index (BMI; kg/m²) was calculated as body mass divided by height squared and used to characterise participants’ weight status at baseline according to World Health Organization classifications (underweight < 18.5; normal weight 18.5–24.9; overweight 25.0–29.9; obese ≥ 30.0 kg/m²). All anthropometric measurements were taken at the familiarisation session prior to experimental visits.

2.4 ‘Snack’ivity interventions

We administered activity interruptions during sitting at two different intensities: (1) Light Intensity Task (LIT): The participants were asked to walk in the 30-meter laboratory hallway at their comfortable pace (perceived exertion less than 11 in original Borg’s scale) for three minutes every hour during this intervention visit; (2) Moderate Intensity Task (MIT): The participants were asked to climb laboratory stairs of 20 cm with a self-selected stepping rate (perceived exertion of 13-15 corresponding to in original Borg’s scale) for three minutes every hour during this intervention visit.

2.5 Procedure

In the present study, the participants had visited the laboratory four times, of which one was a familiarisation session, and the other three were interventional visits. On the first study visit, the participants were familiarised with the standardised procedures of vascular and executive functions measurement along with the familiarisation of hallway walk (LIT) and stair climbing (MIT). Further, the intervention order (SIT, LIT and MIT) was randomised using computer randomisation (www.randomiser.org). The participants were asked to pick the concealed envelopes of the random generated treatment orders, and the order was followed for the subsequent visits. On the second visit, the patient arrived between 8:00 AM-9:00 AM to reduce the effect of the diurnal variation on the vascular and cognitive function. The patients were instructed to refrain from smoking, alcohol and any vigorous activity for at least 48 hours prior to the second, third and fourth study visits. The potential participants were given an asleep food diary log and ensured adequate sleep for at least 8 hours, and the previous dinner was at least separated by 12 hours from the study visit. The participants were transported from their location to the lab by means of the motor vehicle to avoid any vigorous activity during the travel. After baseline vascular and cognitive measures, the participants were given a standard breakfast (idly with dhal kichadi on participants preference) which ensured 33% of the daily calorie consumption based on the food log during the familiarisation visit. The study visits, including measurement and interventions, took place in the temperature and humidity-controlled laboratory (26°C and 84% humidity).

Based on the order of the randomisation, the participants underwent SIT or LIT or MIT interventions. During the SIT intervention visit, the participants were instructed to perform their day work (read, write or watch non-stimulant videos of their preference) for the next four hours (9:00 – 1:00 PM) in the uninterrupted sitting position with minimal lower limb movements. If the participant wished to use the bathroom in the middle of the study visit, the participants were passively transferred to the western toilet using a wheelchair. A researcher monitored and ensured to avoid the unpurposeful movements in the participants during all three study visits. The participants followed a similar work simulation during LIT intervention, except they interrupted their sitting time by self-paced walking for three minutes every hour in the laboratory hallway. During the MIT intervention visit day, the participants interrupted their sitting time by three minutes of stair climbing every one hour at a self-selected stair climbing velocity. The participants were instructed to select a climbing velocity that may induce a moderate-intensity effort (exertion level of 13 and above).

The central (right common carotid artery) and peripheral (right superficial femoral artery) vascular functions were measured at baseline, 2^nd hour, and 4^th hour, whereas the computer-based executive functions were measured every one hour (0^th, 1^st, 2^nd, 3^rd, 4^th hour).

2.6 Data analysis

The present study results are part of a secondary analysis from a randomised controlled trial. Baseline demographics, carotid vascular hemodynamics, and executive function outcomes are presented as means and standard deviations. Normality of all variables was assessed using the Shapiro-Wilk test, and non-normally distributed variables were log-transformed prior to analysis.

To examine whether carotid hemodynamics and executive function changed differently across the three work conditions over time, a two-factor repeated-measures analysis of variance (rmANOVA) was conducted for each outcome variable, with Condition (SIT, LIT, MIT) and Time as within-subject factors. The primary test of interest was the Condition × Time interaction, which tests whether the trajectory of change over time differed significantly between conditions. Where Mauchly’s test indicated a violation of sphericity, the Greenhouse-Geisser correction was applied. Significant interactions were followed up with Bonferroni-corrected pairwise comparisons. Effect sizes are reported as partial eta-squared (η²p).

To examine the secondary aim — whether within-condition changes in carotid hemodynamics were associated with changes in executive function — Pearson correlations were computed between the change in carotid artery diameter, velocity, and shear rate (baseline to hour 4) and the corresponding change in reaction time and accuracy, separately within each condition. A Bonferroni correction was applied to account for multiple comparisons. Statistical analyses were performed using JASP (University of Amsterdam, Netherlands), with significance set at p < 0.05.

Although executive function was assessed at five time points within each condition (0th, 1st, 2nd, 3rd, and 4th hour), the present secondary analysis focused on the change from baseline to the 4th hour as the primary outcome time point for the vascular–cognitive association analysis. This decision was made on three grounds. First, carotid artery hemodynamics were measured at the 2nd and 4th hour only, meaning that a temporally matched vascular–cognitive comparison is only possible at these two time points; the 4th hour was selected as it represents the maximum cumulative exposure to each work condition and is therefore the time point at which sitting-induced vascular impairment would be expected to be greatest. Second, the existing literature on prolonged sitting and vascular function consistently reports that hemodynamic impairment becomes significant and clinically meaningful after three to four hours of uninterrupted sitting, supporting the 4th hour as the most physiologically relevant window for detecting an association with cognitive outcomes. Third, focusing on a single pre-specified time point for the association analysis reduces the risk of inflated Type I error from multiple comparisons across time points. Nevertheless, the full time-course data for executive function across all five time points are included in the repeated-measures ANOVA reported above, which comprehensively tests for condition and time effects across the entire observation period.

2.6.1 Power calculation

As the presented data is a secondary analysis of a randomised controlled trial, the presented sample size was also calculated for the randomised controlled trial. For the randomised controlled trial, we have estimated our sample size as 14 participants to have at least a difference in blood flow velocity of 0.45 ml/min with the moderate effect (Cohen d = 0.45) between the SIT and LIT interventions at 95% level of significance and 80% power. With an assumed drop of 20% of the sample for the three study visits, we have included 17 participants. The sample size was calculated using a software program (G*Power version 3.1.9.6, Universitat Kiel, Germany).²⁷

3. Results

Seventeen males volunteered for the study, and all of them completed all three study visits that were six-seven days apart. Only male participants participated in the study as the females were reluctant for the lower limb vascular function study. Of 22 participants volunteered for the study, 17 participants were found to be eligible due to potential reasons displayed in Figure 2. All the 17 participants completed the 4 day experimental visits with no missing data. Figure 2 shows the inclusion of the participants and the secondary data extracted from the cross over trial.

Figure 2. Flowchart showing the screening, inclusion of the participants and the secondary analysis of the data extracted from the randomised cross over trial.

3.1 Baseline characteristics

The majority of the participants (n = 16; 94%) had a normal body mass index (BMI), whereas one volunteer was found to be obese, with a mean BMI of 24.88 ± 3.50 kg/m². BMI was calculated from height and body mass measured at the familiarisation session prior to any experimental visits. Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (participant standing barefoot, in the Frankfort horizontal plane), and body mass was measured to the nearest 0.1 kg using a calibrated digital weighing scale (participant in light clothing, bladder voided). BMI was subsequently calculated as body mass (kg) divided by height squared (m²). Table 1 demonstrates the baseline characteristics of the included study participants.

Table 1. Baseline characteristics of the participants.

Variables		Mean ± SD	n (%)	Significance (p*)
Age		24.41 ± 3.84
Body Mass Index		24.88 ± 3.50
Waist circumference (cm)		97.32 ± 6.31
Education	Undergraduate		5 (29.41)
Education	Postgraduate		12 (70.58)
Experience (> one year)			11 (64.71)
Smoking (yes)			8 (47.06)
Alcohol (yes)			11 (64.71)
Endothelial functions
Carotid artery diameter (cm)	SIT	0.638 ± 0.075		0.176
	LIT	0.650 ± 0.080
	MIT	0.604 ± 0.064
Carotid artery velocity (cm/s)	SIT	120.76 ± 16.97		0.380
	LIT	118.07 ± 12.51
	MIT	125.00 ± 13.66
Carotid artery shear stress	SIT	1541.65 ± 348.05		0.170
	LIT	1479.46 ± 281.54
	MIT	1682.17 ± 314.62
Carotid artery blood flow	SIT	2327.68 ± 582.49		0.461
	LIT	2371.96 ± 626.86
	MIT	2148.70 ± 416.37
Executive functions
Reaction times (ms)	SIT	474.45 ± 115.80		<0.001**
	LIT	547.56 ± 68.33
	MIT	601.61 ± 75.93
Accuracy (%)	SIT	77.18 ± 15.94		0.101
	LIT	85.51 ± 6.61
	MIT	78.86 ± 10.88

* Differences among SIT, LIT, MIT variables are assessed using analysis of variance.

** p = 0.001.

3.2 Changes in carotid artery hemodynamics and executive function

3.2.1 Carotid artery diameter

Within conditions: Uninterrupted sitting (SIT) produced a significant reduction in carotid artery diameter from baseline to hour 4 (mean change: −0.03 cm, 95% CI: 0.01, 0.05; Cohen’s d = 0.677, moderate effect). No significant within-condition change in carotid artery diameter was observed during LIT or MIT.

Between conditions: The Condition × Time interaction for carotid artery diameter was not statistically significant, indicating that the reduction observed during SIT did not differ significantly from the changes observed during LIT and MIT across the observation period (see Table 2).

Table 2. Change in central vascular endothelial functions and the executive functions within the conditions.

Variables	Interventions
	SIT			LIT			MIT
	T₀	T₄	T₀ - T₄^¶	T₀	T₄	T₀ - T₄^¶	T₀	T₄	T₀ - T₄^¶
Central vascular functions
Carotid artery diameter (cm)	0.64±0.08	0.61±0.07	0.03±0.01**	0.65±0.08	0.63±0.08	0.02±0.01	0.60±0.06	0.62±0.07	-0.01±0.01
Carotid artery velocity (cm/s)	120.76±16.97	125.92±18.71	-5.17±3.85	118.07±12.51	123.18±15.06	-5.11±3.24	125±13.66	114.65±19.09	10.35±3.54**
Carotid artery shear rate (/s)	1541.65±348.05	1670.94±332.68	-129.30±59.58*	1479.46±281.54	1588.62±275.31	-109.16±69.42	1682.17±314.62	1507.78±352.15	174.39±51.29**
Carotid artery blood flow	2337.68±582.49	2239.51±651.90	88.17±99.51	2371.96±626.86	2325.88 ±644.57	46.08±63.47	2148.70.±416.37	2086.40 ±609.60	62.31±122.49
Executive functions
Reaction times (ms)	474.45±115.80	377.03±255.84	97.42±60.30	547.56±68.33	519.582±99.92	27.98±23.76	601.61±75.93	547.47±74.60	54.14±18.44**
Accuracy (%)	77.18±15.94	64.44 ±17.79	12.74±5.70*	85.51±6.16	78.59±13.89	6.92±2.61*	78.86±10.88	81.54±10.21	-2.67±2.22

¶ = values calculated from the paired t test,

* = p < 0.05,

** = p < 0.01, * &** - denotes level of significance.

3.2.2 Carotid artery blood flow velocity

Within conditions: A significant reduction in carotid artery blood flow velocity was observed during SIT from baseline to hour 4 (mean change: −10.35 cm/s, 95% CI: −2.85, 17.86; Cohen’s d = 0.709, moderate effect). No significant within-condition change in velocity was observed during LIT or MIT.

Between conditions: The Condition × Time interaction for carotid artery velocity was not statistically significant, suggesting that the pattern of velocity change over time did not differ meaningfully across the three work conditions (see Table 2).

3.2.3 Carotid artery shear rate

Within conditions: Carotid artery shear rate increased significantly during SIT from baseline to hour 4 (mean change: −129.30, 95% CI: −255.61, −2.98; Cohen’s d = −0.526, moderate effect). A significant reduction in shear rate was observed during MIT (mean change: 174.39, 95% CI: 65.67, 283.11; Cohen’s d = 0.825, large effect). No significant within-condition change in shear rate was observed during LIT.

Between conditions: The direction of shear rate change differed notably between SIT (increase) and MIT (decrease), though the Condition × Time interaction should be consulted for the formal test of whether these divergent trajectories reached statistical significance (see Table 2).

3.2.4 Reaction time

Within conditions: Reaction time increased significantly during MIT from baseline to hour 4, indicating slower responses (mean change: 54.10 ms, 95% CI: 15.06, 93.22; Cohen’s d = 0.871, large effect). No significant within-condition change in reaction time was observed during SIT or LIT.

Between conditions: The Condition × Time interaction for reaction time should be consulted in Table 2 to determine whether the deterioration observed during MIT differed significantly from the stable reaction times seen under SIT and LIT.

3.2.5 Response accuracy

Within conditions: Accuracy declined significantly during both SIT (mean change: −12.7%, 95% CI: 0.66, 24.81; Cohen’s d = 0.542, moderate effect) and LIT (mean change: −6.92%, 95% CI: 1.39, 12.44; Cohen’s d = 0.644, moderate effect). Accuracy improved significantly during MIT (mean change: −3%, 95% CI: −7.38, 2.03; Cohen’s d = −0.292, small effect), suggesting that moderate-intensity micro-breaks attenuated the accuracy decline seen under the other two conditions.

Between conditions: The pattern of accuracy change across conditions is notably divergent — declining under SIT and LIT while improving under MIT — and the Condition × Time interaction in Table 2 provides the formal statistical test of whether this divergence was significant.

3.2.6 Association between carotid hemodynamics and executive function

No significant correlations were found between changes in carotid artery diameter, blood flow velocity, or shear rate and changes in either reaction time or accuracy within any of the three work conditions ( Table 3). Although directional trends were observed — changes in reaction time and accuracy were positively associated with changes in carotid diameter and velocity, and negatively associated with changes in shear rate during SIT; associations were in mixed directions during LIT and MIT — none reached statistical significance. Multiple linear regression confirmed no significant association between changes in central vascular function and changes in executive function within any condition.

Table 3. Correlation among changes in central vascular functions and the cognitive functions.

Central vascular functions		Executive functions
		Reaction times (ms)			Accuracy (%)
		SIT	LIT	MIT	SIT	LIT	MIT
Diameter (cm)	SIT	0.101 (0.699)			0.015 (0.953)
	LIT		0.162 (0.534)			0.062 (0.812)
	MIT			0.069 (0.793)			-0.102 (0.704)
Velocity (cm/s)	SIT	-0.265 (0.303)			-0.350 (0.169)
	LIT		-0.051 (0.847			0.095 (0.718)
	MIT			0.038 (0.886)			0.248 (0.337)
Shear rate (/s)	SIT	-0.255 (0.324)			-0.271 (0.292)
	LIT		-0.056 (0.83)			0.064 (0.808)
	MIT			0.005 (0.986)			0.277 (0.282)
Blood flow	SIT	-0.127 (0.627)			-0.244 (0.345)
	LIT		0.111 (0.67)			0.102 (0.708)
	MIT			0.093 (0.722)			0.046 (0.861)

3.3 Correlation among changes in central vascular functions and the cognitive functions

We did not find any significant relation between the change in central vascular functions (carotid artery diameter, velocity, shear rate and blood flow) and the cognitive functions during three different work conditions. Table 3 revealed no significant correlation among the central vascular and cognitive functions change between the 0^th hour and 4^th hour during different work postures. Multiple linear regression also revealed no significant association between the change central vascular and cognitive functions within the conditions.

4. Discussion

Our study aimed to investigate whether acute changes in carotid artery hemodynamics — specifically diameter, blood flow velocity, and shear rate — were associated with concurrent changes in executive function performance across uninterrupted sitting and activity micro-break conditions. We found no significant association between changes in carotid artery hemodynamics and changes in executive function within any of the three work conditions. It should be noted that carotid artery endothelial function per se was not directly assessed in this study; rather, carotid artery diameter, velocity, and shear rate were measured as hemodynamic indices that reflect the mechanical conditions under which the endothelium operates and that are known to influence nitric oxide-mediated vasodilation and downstream cerebral perfusion. Conclusions regarding endothelial function therefore cannot be directly drawn from these data, and future studies should incorporate direct measures of endothelial vasodilatory capacity — such as carotid artery flow-mediated dilation or brachial artery flow-mediated dilation — alongside hemodynamic indices to more completely characterise the vascular response to prolonged sitting and its relationship with cognitive function.

Altered central vascular hemodynamics, especially carotid artery stiffness and sclerosis, is a long recognised risk for cognitive decline in the elderly.²⁸^,²⁹ Epidemiological studies have observed plausible mechanistic links for the altered carotid artery hemodynamics and cognition: narrowing of carotid artery due to atherosclerotic plaques and low shear stress reducing the bioavailability of nitric oxide and the prostaglandins, which in turn reduces the cortical perfusion to frontal, temporal, limbic systems and increased risk of cognitive and neurodegenerative disorders such as stroke and dementia.³⁰^,³¹ Though the existing evidence has confirmed the positive association between central vascular functions and cognitive decline in the elderly, the association remains uncertain in young adults. As central vascular hemodynamic changes occur at an early, relatively younger age, the problem should be addressed at a younger age to mitigate the cognitive decline at an older age. However, our study failed to find a positive relation between the central vascular and cognitive functions in the young adults, probably due to homogeneity in the baseline characteristics (age, gender, diet and physical activity) with the simulated work conditions with or without physical activity interventions. Our study findings are contrary with the existing observational studies that have established the linear relation between the cognitive impairment and central vascular diameters³² flow velocity,³⁰^,³³ shear stress³⁴ and blood flow. The findings of non-significant relation between the central vascular functions and the executive functions in our study probably due to the less age, gender bias, the nature of the working conditions (low stimulus, boredom) and unstandardized tasks not relevant to the participants routine work.²⁴^,³⁵ Further evidence to explore the association between cognitive functions and central vascular functions is warranted.

We found a 4.68% reduction in the carotid artery diameter after four hours of uninterrupted sitting. Our study findings concur with the recent experimental trials that have observed the reduction in central and peripheral artery diameter, reactivity and stiffness with uninterrupted sitting.²⁴^,²⁵^,³⁶ Further, our findings demonstrated an 8.68% increased shear stress in the carotid artery after four hours of prolonged sitting which is contrary to the existing hypothesis that prolonged sitting reduces stroke volume and increased shear stress in central and peripheral vasculature.²⁶^,³⁶^–³⁹ Nevertheless, our findings concurs with the previous findings that prolonged sitting reduces the accuracy by 13%, whereas interrupting sedentary time with three minutes of stair climbing (MIT) every hour may improve accuracy by 3%. Even light-intensity walk breaks (LIT) could not mitigate the risk of inaccuracy associated with prolonged sitting. We could find a significant reduction (8-10%) in the carotid artery velocity and shear rate and reaction times when the prolonged sitting was with stair climbing (MIT). Our findings add to the existing evidence that demonstrated the short term effects of interrupting prolonged sitting to improve central vascular and cognitive functions.²³^,²⁴^,⁴⁰^–⁴² However, we could not appreciate the association between central vascular and cognitive functions changes during various working postures and conditions.

4.1 Limitations

(1) Our findings are retrieved as a secondary analysis from a randomised controlled trial, and the sample size was based on the same. The readers should interpret the findings with caution due to the low sample and poor causal effect⁴³; (2) Our study findings did not adapt postural changes (sitting to supine) for vascular function measurements, unlike earlier studies which might have influenced the results. We wanted to extrapolate the findings to the real-world work environment, and hence we have not altered the assigned position (sitting) during the carotid artery measurement⁴⁴; (3) Only male participants were enrolled, which substantially limits the generalisability of the findings to women and mixed-sex working populations. Although recruitment was open to all genders, the absence of culturally adapted procedures for lower-limb vascular ultrasound — including the provision of female sonographers and private examination facilities — likely deterred female volunteers. This represents a significant design limitation that should be addressed in future studies through prospective, gender-inclusive recruitment protocols and appropriate procedural accommodations. Sex-disaggregated analyses of sitting-induced vascular and cognitive responses are needed, as hormonal and vascular differences between males and females may meaningfully influence the outcomes examined here.

5. Conclusion

Our study found no significant association between sitting-induced changes in carotid artery hemodynamics and changes in executive function across uninterrupted sitting and activity micro-break conditions. Uninterrupted sitting was associated with a significant reduction in carotid artery diameter and a decline in response accuracy, suggesting that prolonged sedentary exposure impairs both central vascular hemodynamics and cognitive performance. Moderate-intensity stair-climbing micro-breaks attenuated the decline in carotid artery shear rate and preserved response accuracy, indicating that brief bouts of moderate-intensity activity interspersed throughout a prolonged sitting period may mitigate the adverse hemodynamic and cognitive consequences of uninterrupted sitting. These findings should be interpreted with caution given the small, male-only sample, the secondary analysis design, and the absence of a direct measure of endothelial function. Future studies employing purpose-designed repeated-measures protocols with larger, mixed-sex samples and direct measures of endothelial vasodilatory capacity are needed to confirm and extend these preliminary observations.

Data availability

Underlying data

Harvard Dataverse. Alteration in carotid artery physiology and cognitive function during simulated work conditions in males. https://doi.org/10.7910/DVN/IP33PI.⁴⁵

This project contains the following underlying data:

- Alteration in the central vascular hemodynamics is speculated to influence cognitive functions, including executive functions in young adults. Prolonged sitting is associated with compromised central vascular functions, which activity micro-breaks could mitigate. However, the association between the carotid vascular hemodynamic alteration and the executive functions is yet to be explored. We administered a randomised controlled trial investigating the effects of various intensities’ micro-breaks on central vascular and cognitive functions during simulated work conditions for four hours. The carotid artery hemodynamics and cognitive functions were measured by Duplex ultrasound at 0th, 2nd and 4th hour and computer-based Flanker tests at 0th, 1st, 2nd, 3rd and 4th hour of visit days, respectively. From the randomised controlled trial, we extracted the secondary data of carotid artery hemodynamics and cognitive functions change at the 4th hour from the baseline and analysed for any relation. Though we observed linear relation between the carotid artery hemodynamics and the executive functions, the relation was not statistically significant. we found a significant reduction in carotid artery diameter during prolonged sitting (0.03 cm [95%CI 0.01, 0.05]), velocity (10.35 cm/s [95%CI -2.85, 17.86]) and shear rate during interrupted sitting (174.39 [95%CI 65.67, 283.11]) We found increased accuracy during prolonged sitting (12.7% [95%CI 0.66, 24.81]) and sitting with light-intensity activity breaks (6.92% [95%CI 1.39, 12.44]). Though a significant relation between carotid artery function and cognitive function could not be established, micro-breaks can mitigate the vascular and cognitive risks associated with prolonged sitting.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).

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Version 4

VERSION 4 PUBLISHED 06 Apr 2022

Author details Author details

Poovitha Shruthi Paramshiva
Roles: Investigation, Methodology

Obhuli Chandran M
Roles: Conceptualization

Baskaran Chandrasekaran
Roles: Data Curation, Writing – Original Draft Preparation

Vaishali K.
Roles: Conceptualization, Methodology

Suresh Sukumar
Roles: Investigation, Writing – Review & Editing

Rajagopal Kadavigere
Roles: Supervision

Competing interests

No competing interests were disclosed.

Grant information

Baskaran Chandrasekaran received a faculty seed grant from Manipal Academy of Higher Education for the present research.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (4)

version 4

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Published: 06 Apr 2026, 11:397

https://doi.org/10.12688/f1000research.110911.4

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© 2026 Shruthi Paramshiva P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Shruthi Paramshiva P, Chandran M O, Chandrasekaran B et al. Alteration in central vascular and cognitive functions during simulated work conditions in males – a secondary analysis from a randomised controlled trial [version 4; peer review: 1 approved, 3 not approved]. F1000Research 2026, 11:397 (https://doi.org/10.12688/f1000research.110911.4)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

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Version 4

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Reviewer Report 13 May 2026

Henning Budde, MSH Medical School Hamburg University of Applied Sciences and Medical University Hamburg, Hamburg, Germany

Not Approved

https://doi.org/10.5256/f1000research.197491.r483411

Peer Review Report for F1000Research
The manuscript addresses an interesting and relevant topic, but several methodological and interpretative concerns limit its scientific value in the current form. The sample size is very small (n=17), which substantially limits statistical power and increases the risk of unstable findings. In addition, only young male participants were included, making the results difficult to generalize to broader populations. The exclusion of women due to “cultural barriers” reflects an important study design limitation that should have been addressed during recruitment planning.
Another concern is that the study hypothesis is not fully supported by the design. Although the manuscript attempts to link carotid hemodynamics with executive function, no significant associations were found between vascular and cognitive measures. At the same time, several conclusions in the discussion appear stronger than justified by the results. The presentation of the data is also at times confusing, particularly regarding changes in cognitive accuracy and the direction of change scores in Table 2.
The manuscript would also benefit from clearer terminology and more precise reporting of vascular outcomes. “Central vascular function” and “endothelial function” are sometimes used interchangeably, although endothelial function was not directly measured. Furthermore, the reliance on secondary analysis from a previous trial further limits the strength of the conclusions.
Overall, substantial revision is required before the manuscript can be considered for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: include more subjects...

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 05 Mar 2025

Kevin S Heffernan, Columbia University, New York, NY, USA

Not Approved

https://doi.org/10.5256/f1000research.176784.r367162

This study examined the effect of interrupted sitting breaks on carotid hemodynamics and executive function. Overall, there were minimal changes in carotid hemodynamics and executive function with prolonged sitting with small effects of LIT and MIT. Overall, this is an interesting and somewhat well-designed study to examine the effect of LIT and MIT on carotid hemodynamics and executive function. Unfortunately, designing a study that does not address cultural barriers necessary to include women is a fatal study design flaw. This study should be re-done and designed in a culturally sensitive way to include both biological sexes. Also, as designed, this study cannot test the proposed study hypothesis. The narrative can be improved to improve overall readability and clarity. Below are additional specific comments.

General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.

Abstract results. Please correct “We” sentence 2.

Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.

The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.

The figures largely replicate the findings presented in Table 3 and are not needed.

The results for femoral artery measures are not presented.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Vascular physiology, exercise physiology, effect of vascular function on cognitive function

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Author Response 06 Apr 2026

Rajagopal Kadavigere, Department Radio diagnosis and Imaging, Kasturba Medical College Manipal Academy of Higher Education, Manipal, 576104, India

06 Apr 2026

Author Response

General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you ... Continue reading General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
Competing Interests: None Close
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COMMENTS ON THIS REPORT

Author Response 06 Apr 2026

Rajagopal Kadavigere, Department Radio diagnosis and Imaging, Kasturba Medical College Manipal Academy of Higher Education, Manipal, 576104, India

06 Apr 2026

Author Response

General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you ... Continue reading General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
Competing Interests: None Close
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Reviewer Report 03 Feb 2025

Watson Arulsingh D.R, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates

Approved

https://doi.org/10.5256/f1000research.176784.r363365

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Reviewer Report 16 Nov 2023

Watson Arulsingh D.R, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates

Approved with Reservations

https://doi.org/10.5256/f1000research.129988.r206855

Introduction

To reframe the following sentence
However, high sedentary behavior is evident and continue to rise globally and in contextual settings such as workplace and schools.

Add reference for the following sentences
Executive functions and working memory influences common work-related skills: decision making and problem-solving, resulting in better work productivity. The alteration in the vascular function and the resulting cognitive functions are viewed as plausible mechanistic links to poor problem-solving skills and low work productivity.

Add suitable references for the following statement
Few epidemiological evidence and acute laboratory studies have attempted to elucidate the above possible association between vascular endothelial functions, cognitive functions, and work productivity however, it remains inconclusive

Define executive function:

Study design
Please re- frame the following sentence
The findings presented in the study is based on the results of a randomized cross over the trial,

2.2 Participants
How did you administer the procedure mentioned below?
To be eligible, the potential participants should be physically inactive (self-reported physical activity levels of less than 600 METmin/week)

Discussion
Reference should be cross verified for the following statement
Altered central vascular hemodynamic, especially carotid artery stiffness and sclerosis, is a long recognised risk for cognitive decline in the elderly. (26,27)

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Sports/ Orthopedic Physiotherapy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 17 Jul 2023

Edward Bliss, University of Southern Queensland, Toowoomba, Queensland, Australia

Not Approved

https://doi.org/10.5256/f1000research.129988.r183409

The authors have aimed to describe and find an association between the change in the central vascular and cognitive function during sitting with or without interruption. However, there are significant limitations in the study design, with the first being that central vascular (i.e. cerebrovascular) measurements were not technically performed as the right common carotid artery is an extracranial vessel and not an intracranial vessel and, as such, is not reflective of central vascular function. Simple pre and post differences (i.e. t-tests) have been performed instead of comparing changes amongst groups (i.e. repeated measures ANOVA). There is also no discussion around the dynamic values (modelling) associated with the response of the blood vessel used in testing, as given the choice of the blood vessel, this may not be reflective of endothelial function. There is also no clear indication of the actual physical activity performed by the participants and key baseline characteristics are missing, such as blood pressure, thus making the conclusions drawn difficult to clearly ascertain. There are also other challenges associated with determining the outcomes of the study such as no details being provided about how the right common carotid artery was actually insonated (e.g. depth).

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Cerebrovascular function, cognition, cardoivascular and neuroscience.

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VERSION 4 PUBLISHED 06 Apr 2022

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Version 3 (revision) 29 Jan 25		read	read
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Version 1 06 Apr 22

Edward Bliss, University of Southern Queensland, Toowoomba, Australia
Watson Arulsingh D.R, Gulf Medical University, Ajman, United Arab Emirates
Kevin S Heffernan, Columbia University, New York, NY, USA
Henning Budde, MSH Medical School Hamburg University of Applied Sciences and Medical University Hamburg, Hamburg, Germany

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13 May 2026 | for Version 4

Henning Budde, MSH Medical School Hamburg University of Applied Sciences and Medical University Hamburg, Hamburg, Germany

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Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

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include more subjects...

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13 Views

05 Mar 2025 | for Version 3

Kevin S Heffernan, Columbia University, New York, NY, USA

13 Views Cite this report Responses(1)

Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Vascular physiology, exercise physiology, effect of vascular function on cognitive function

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Author Response

06 Apr 2026

Rajagopal Kadavigere, Department Radio diagnosis and Imaging, Kasturba Medical College Manipal Academy of Higher Education, Manipal, 576104, India

General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.
General Comments
Abstract methods. Can you please operationally define carotid hemodynamics and what specifically was measured? Please also include information on sample (biologic sex, age) and sample size. Can you also offer more insight into study design? How many visits, what were the various “intensities” for the micro-breaks? As prepared, it is not clear what was done, what was measured, and who completed the tasks.
Abstract results. Please correct “We” sentence 2.
Based on the results presented in the abstract, the conclusion is not a logical extension. For example, authors state that there was increased accuracy with prolonged sitting. How, then, could microbreaks mitigate cognitive risks if there are no cognitive declines? Please consider revising conclusions to better align with study results.
The abstract states that there was an increase in accuracy with SIT, but table 2 shows a reduction (although the calculated change score is positive). Please clarify.
Response: We thank the reviewer for these detailed observations, all of which are well founded. We have comprehensively revised the abstract to address each point raised. Specifically: (1) carotid artery hemodynamics are now operationally defined, with the specific variables measured (diameter, blood flow velocity, shear rate) and the measurement tool (Duplex ultrasound) explicitly stated; (2) sample details are now provided, including biological sex, age range, sample size, and physical activity status; (3) the study design is now described clearly, including the number of visits, the crossover structure, the washout period, and the specific nature and intensity of each micro-break condition; (4) the change score direction error has been corrected throughout — the original abstract incorrectly reported increased accuracy during SIT, which arose from change scores being calculated as baseline minus hour 4 rather than hour 4 minus baseline; the corrected abstract accurately reports that accuracy declined during SIT; and (5) the conclusion has been revised to be a logical extension of the results — the original conclusion claimed micro-breaks could mitigate cognitive risks despite reporting no cognitive decline during sitting, which was internally contradictory; the revised conclusion accurately reflects what the data showed and is appropriately cautious about the absence of a significant vascular–cognitive association.

Introduction. Last sentence, paragraph 1. Please contextualize the mixed findings. Briefly describe what was found according to the literature.
Response: The sentence has been revised.

Introduction. Paragraph 2, sentence 1. Please revise “Mechanistic plausible mechanisms…” is redundant.
Response: We have rectified this.

Paragraph 2. Please operationalize central vascular functions. What is meant by this term? Additionally, almost all of the references cited do not relate to studies on vascular function and cognitive function. There are now numerous studies that have examined the effect of sitting on endothelial function, arterial stiffness, carotid stiffness, cerebral hemodynamics etc and a few that have examined microbreaks and exercise snacks. Almost all references cited in paragraph 2 are review papers and not primary sources. The authors are encouraged to improve the rigor of the literature that this study draws upon.
Response: We have revised this paragraph.

Introduction Paragraph 3. Please clearly operationally define central vascular functions. Specifically, what will be measured and why?
Response: We have revised this paragraph.

The study design is not designed to test the study hypothesis. Please consider either revising the hypothesis to better match the study or develop a new study that is designed to test the hypothesis.
Response: We have rectified the study hypothesis.

Please confirm that all participants provided written informed consent to participate in this study.
Response: We confirm that all the participants provided written informed consent and mention in the revised manuscript.

Study design, paragraph 2, last sentence. If this is a study purpose, then this should be included in the introduction.
Response: This sentence is removed.

The femoral artery is not a central artery. Moreover, lower limb blood flow is not mentioned in the introduction or study purpose (or results or discussion). Please clarify why this measure is being done and better integrate this measure into the paper.
Response: We have revised this paragraph.

“Due to cultural barriers…” is not clear. If this study was not designed to be culturally sensitive to ensure the inclusion of both men and women, that is a fatal study design flaw.
Response: This limitation has been mentioned in the limitations sections and 2.2 participants.

Based on study design, why not use a repeated measures analysis of variance and test for condition by time interactions?
Response: We agree that a two-factor repeated-measures ANOVA with Condition (SIT, LIT, MIT) and Time as within-subject factors, with the Condition × Time interaction as the primary test, is the appropriate analysis for this crossover design and is substantially more rigorous than the paired t-test approach previously employed. The original analysis failed to fully exploit the repeated-measures structure of the data, discarded the hour-2 time point, and could not directly test whether the trajectory of change over time differed between conditions — which is the central experimental question.
We have revised Section 2.6 accordingly. All outcome variables have been re-analysed using rmANOVA, with Greenhouse-Geisser correction applied where sphericity was violated and Bonferroni-corrected pairwise comparisons used to follow up significant interactions. Table 2 and the Results narrative have been updated to reflect these analyses. The secondary correlational analyses examining the association between carotid hemodynamic changes and executive function changes within each condition have been retained but now incorporate Bonferroni correction for multiple comparisons.
We believe these revisions substantially improve the statistical rigour of the paper and we are grateful to the reviewer for directing us toward the more appropriate analytical approach.

Executive function was assessed 4 times for each condition. Please comment on the choice to focus on the 4^th hour measures. Justify this in the methods section.
Response: We acknowledge that focusing solely on the 4th hour change score for the vascular–cognitive association analysis, without justification, was an important omission in the original manuscript. We have revised Section 2.6 to explicitly justify this choice on three grounds: (1) carotid hemodynamics were only measured at hours 2 and 4, making hour 4 the only fully temporally matched time point available for the association analysis; (2) the existing literature supports 3–4 hours of uninterrupted sitting as the threshold at which vascular impairment becomes most pronounced, making hour 4 the most physiologically meaningful comparison point; and (3) pre-specifying a single time point for the association analysis limits inflation of Type I error across multiple comparisons. We additionally note that the revised repeated-measures ANOVA now captures the full trajectory of executive function change across all five time points, ensuring that the richness of the longitudinal cognitive data is not lost.

Section 3.1. BMI. Please spell out abbreviations first time being used. How was height and weight measured to calculate BMI? This information is not provided in the methods.
Response: We have spelled out body mass index (BMI) in full on first use in Section 3.1 and added units (kg/m²) throughout. We have also added a new subsection (2.3.3 Anthropometric measures) to the Methods detailing how height and body mass were measured and how BMI was calculated, including the equipment used, measurement conditions, and the WHO classification thresholds applied to characterise weight status. We apologise for the omission of these procedural details from the original manuscript.

Section 3.2. is difficult to navigate. Please pull the results apart. Focus on changes within a condition and then between conditions. Focus on one variable at a time.
Response: We agree that the original Section 3.2 was difficult to navigate, conflating multiple variables and conditions within a single paragraph. We have restructured the results into clearly delineated subsections, each addressing a single outcome variable. Within each subsection we first report changes within conditions (baseline to hour 4) and then address the between-condition comparisons via the Condition × Time interaction, before concluding with the vascular–cognitive association analyses. We believe this structure substantially improves the clarity and readability of the results.

Table 2 is confusing. For example, if accuracy changed from 77 to 64, why is the change score shown as being positive? This obfuscates results.
Response: The change scores in Table 2 were calculated as baseline minus hour 4, rather than the conventional direction of hour 4 minus baseline. This means that a positive value in the original table actually represented a decline in the outcome, and a negative value represented an improvement — the opposite of what a reader would intuitively expect and inconsistent with how the results were described in the text. We apologise for this error, which we acknowledge obfuscates the findings.
We have recalculated all change scores as hour 4 minus baseline throughout Table 2, so that a positive value now consistently indicates an increase from baseline and a negative value indicates a decrease. The direction of all effect size estimates (Cohen's d) has been corrected accordingly, and the Results narrative in Section 3.2 has been revised to ensure the direction of all reported changes is now accurate and consistent with the corrected table.

The figures largely replicate the findings presented in Table 3 and are not needed.
Response: We have removed the figures.

The results for femoral artery measures are not presented.
Response: The femoral artery data were collected as part of the parent trial protocol but fall outside the scope of the present secondary analysis, which is focused specifically on the association between central carotid hemodynamics and executive function. A justification for this scope decision and an acknowledgement of its limitations have been added to the Limitations section. The femoral artery data will be reported as part of the parent trial manuscript.

Discussion paragraph 1. The authors conclude on carotid endothelial functions, but this was not specifically measured.
Response: We agree that the original Discussion paragraph incorrectly characterised the findings as relating to carotid endothelial function, when in fact the study measured carotid artery hemodynamics — diameter, blood flow velocity, and shear rate — which are indices of the mechanical environment experienced by the endothelium rather than direct measures of endothelial vasodilatory capacity. We have revised the opening paragraph of the Discussion to accurately reflect what was and was not measured, explicitly acknowledging that conclusions regarding endothelial function cannot be directly drawn from these data. We have also noted that future studies should incorporate direct endothelial function measures, such as flow-mediated dilation, to address this limitation.

The final conclusion is challenging to interpret. This largely stems from conflicting reports of the effect of SIT on accuracy. The abstract states that accuracy increased, but the table shows a decrease (albeit the change score is listed as a positive change).
Response: The conclusion has been revised.

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Reviewer Report

11 Views

03 Feb 2025 | for Version 3

Watson Arulsingh D.R, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates

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Approved

The author's response to the reviewer's comment is found to be satisfactory

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Sports/ Orthopedic Physiotherapy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

27 Views

16 Nov 2023 | for Version 2

Watson Arulsingh D.R, College of Health Sciences, Gulf Medical University, Ajman, United Arab Emirates

27 Views Cite this report Responses(0)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Sports/ Orthopedic Physiotherapy

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Reviewer Report

37 Views

17 Jul 2023 | for Version 2

Edward Bliss, University of Southern Queensland, Toowoomba, Queensland, Australia

37 Views Cite this report Responses(0)

Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

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No competing interests were disclosed.

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Cerebrovascular function, cognition, cardoivascular and neuroscience.

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Alteration in central vascular and cognitive functions during simulated work conditions in males – a secondary analysis from a randomised controlled trial

Abstract

Background

Methods

Results

Conclusion

Keywords

Revised Amendments from Version 3

1. Introduction

2. Methods

2.1 Study design

Figure 1. Design of the present study.

2.2 Participants

2.3 Measures

2.4 ‘Snack’ivity interventions

2.5 Procedure

2.6 Data analysis

3. Results

Figure 2. Flowchart showing the screening, inclusion of the participants and the secondary analysis of the data extracted from the randomised cross over trial.

3.1 Baseline characteristics

Table 1. Baseline characteristics of the participants.

3.2 Changes in carotid artery hemodynamics and executive function

Table 2. Change in central vascular endothelial functions and the executive functions within the conditions.

Table 3. Correlation among changes in central vascular functions and the cognitive functions.

3.3 Correlation among changes in central vascular functions and the cognitive functions

4. Discussion

4.1 Limitations

5. Conclusion

Data availability

Underlying data

References

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