This study was approved by the Institutional Review Board of the UPR-MSC Human Research Protection Office (A2540116). Subjects of this study were recruited through advertisements published around the University of Puerto Rico, Medical Sciences Campus and Facebook page of the physical therapy students involved in this study. The study was carried out in the Biomechanics Laboratory of the Doctoral Program in Physical Therapy at the School of Health Professions in the Medical Sciences Campus, University of Puerto Rico (UPR-MSC).

Subjects were invited to participate in the study through word of mouth, flyers and social media announcements during May 1, 2016 to December 15, 2016 for trial and recruitment purposes. Contact information was provided and potential participants called the Principal Investigator (PI) to express their interest in participating and schedule an appointment. Subjects were instructed to assist the biomechanics laboratory at the UPR-MSC. During the scheduled meeting, the PI explained the details of the study and when participants choose to partake there were given the informed consent. They had no specific time to finish reading informed consent, and they were encouraged to ask any questions during that process. When participants communicated to the PI that they finished reading the informed consent, the PI asked questions related to the study to ensured subjects did understand the purpose of the study and their role in the study.

The inclusion criteria for the study were designed to ensure a homogenous sample among the participants. 1) subjects between 21 to 40 years of age, 2) functional flexibility in the lower extremities, 3) functional muscular strength in the lower extremities, 4) completing the AHA/ASCM Health/Fitness Facility Pre-participation Screening Questionnaire, 5) a Body Mass Index of 18.5 to 29.9, 6) arterial blood pressure less than or equal to 140/90 mmHg, 7) pulse at rest between 45–90 beats per minute (BPM), and 8) 95% or more of oxygen saturation (spO2).

The exclusion criteria were designed to identify any factor that could alter postural control, other than energy drinks, or endanger the participant's safety. 1) subjects under the age of 21 and/or over the age of 40, 2) answering any questions affirmative on the pre-participation questionnaire, 3) cardiovascular problems, 4) severe balance problems, 5) taking any sedative or stimulant medications, 6) medical history of any neurological condition, 7) a fall in the last 3 months, 8) having undergone a surgical procedure in the past 6 months, 9) pain in the lower extremities and / or lower back, 10) have suffered a lower back injury and/or in the lower extremities in the last 6 months, 11) is pregnant or suspecting pregnancy, 12) have experienced adverse effects after caffeine consumption, 13) caffeine consumption 12 hours prior to the study intervention, 14) allergies to one of the energy drink ingredients, and 15) people unable to consent.

After signing the informed consent and reviewing the inclusion and exclusion criteria, participants were subjected to a preliminary screening of participation using a list of questions and the AHA / ACSM Health / Fitness Facility Questionnaire ⁹ . This questionnaire's purpose is to ensure the safety of the participants to engage in physical activity by assessing the subjects and family history of cardiovascular diseases. Afterwards, we assessed subjects’ vital signs, blood pressure, pulse, and spO2, to ensure subjects were able to participate in physical activity safely ingest a moderate intake of caffeine without complications. Since postural control is negatively correlated with increased adiposity, weight and height data were measured to obtain a classification according to the Body Mass Index. A range of 18.5 to 29.9 was required to participate ^{10, 11} . During the physical examinations the subjects also performed a Romberg test to rule out any obvious impairment in static balance, a modified Sit and Reach Test for the evaluation of functional flexibility, Sit to Stand Test (30 Seconds) for the assessment of lower limb functional strength, and a Tecumseh Step Test to evaluate the response of the cardiovascular system to a submaximal cardio test ^{12– 15} . After the physical examination, the participants had a rest period between 10–20 minutes before starting the balance tests protocol of (BALT's).

Twenty-three people contacted the researchers to participate in the study (see Supplementary File 2). We excluded 3 subjects from participating in the study for the following: 1) BMI of 34 (male), 2) did not pass pre-participation questionnaire for reporting adverse effects after ingesting caffeine (female) and 3) high blood pressure (male). Of the twenty participants, eleven (55%) were men and 9 (45%) women, with an average of 170.73 pounds, 26.1 years of age and 67.1 inches of height. The caffeine group consisted of 5 (50%) male participants and 5 (50%) female participants, while the energy drink group consisted of 6 (60%) female and 4 (40%) male participants ( Figure 1).

The 20 healthy young subjects selected, were randomly assigned (simple randomization, flipping a coin 3 times, heads was energy drink, tales was caffeine) to control (caffeine) group (n = 10) and experimental (energy drinks) group (n = 10). Vital signs were retaken before the balance test protocol to ensure that the subjects remained within the previously established inclusion values.

Each participant from both groups performed 8 balance tests (BALT's) which alter or cancel, individually or combined, sensory input from the sensory systems involved in postural control ( Table 1). The order of the tests was changed systematically between subjects; therefore, they did not perform the tests in the same order to eliminate external factors such as fatigue or accommodation to the BALT's, which could alter the results. The BALT’s were conducted on the MatScan,™ (TekScan, Boston, MA) a pressure platform containing sensors that measure a displacement of the center of pressure in centimeters square (cm ²) (CoP), anteroposterior (AP), and mediolateral (ML) sway in centimerters (cm) ¹⁶ . The data collected from the pressure mat was analyzed with Tekscan Sway Analysis Module (SAM) software designed for this purpose. The subjects stood for 30 seconds on the pressure platform during each test.

The first 4 BALT’s were carried out by placing the pressure platform on the floor, a stable surface. These tests were: 1) Open Eyes (EO) with a fixed point to evaluate all the systems involved in the postural control (visual, vestibular, somatosensory); 2) Eyes Closed (EC) to evaluate the vestibular and somatosensory system, while eliminating the visual sensory input; 3) Eyes Open while actively moving the head up and down (HUD) to evaluate the visual and somatosensory system, while altering the vestibular system with head movements (EO HUD) (For HUD movements a metronome 2/4 60 BPM was used to maintain a fixed frequency of about one spin per second in motion); 4) Eyes closed and actively moving the head up and down (EC HUD) to assess the effect of removing the visual input, in combination of an alteration of the vestibular system with the head up and down movements.

The remaining 4 BALT’s were the same as the 4 previously mentioned tests with the difference that the subject stood on an unstable surface (foam mat) that was placed on top of the pressure platform to alter the somatosensory (proprioceptive) system. The BALT’s on the unstable surface were: 1) Open Eyes (MAT EO), standing on the unstable surface to evaluate the visual and vestibular system while the somatosensory is altered; 2) Eyes Closed (MAT EO), standing on the unstable surface to evaluate the vestibular system (the somatosensory was modified and the visual system removed); 3) Eyes Open (MAT EO HUD) while actively moving the head up and down to evaluate the visual system, while altering the vestibular and somatosensory system; 4) Eyes Closed (MAT EC HUD) while actively moving the head up and down (in this test all three systems were altered). The same frequency of motion used for BALT’s 3 and 4 in the stable surface (1 spin per second, 60 BPM) was maintained for the BALT’s with HUD movements on the unstable surface.

After the initial 8 pre BALT's, to the experimental group, 160 mg of caffeine was given through one energy drink (16 ounces). Monster energy drink was chosen because, unlike Redbull previously used by Enriquez, the label of Monster Energy drink exhibits the guarana additive, a potential CNS stimulant, in its nutritional label ^{17, 18} . To the control group, we gave a caffeine pill of 200mg, a moderate dose in healthy adults which has been used in numerous studies on different topics and has not been associated with adverse effects such as toxicity, cardiovascular effects, behavior changes, among other things ¹⁸ . Because peak absorption of caffeine is reached within 30–45 minutes after the ingestion of caffeine/energy drink, there was a rest period of 30 minutes ^{19, 20} . Vital signs were measured after the rest period, for the third time . The same 8 BALT’s were then performed, for post ingestion results.

We used the statistical package for the social sciences (SPSS) version 19 for all the data analysis. In this study, we used "Teskcan Sway Analysis Module (SAM)" software for our data collection. For data analysis, we first did a Shapiro-Wilks test to determine normality and to eliminate atypical values, or outliers for the CoP and sway data. Then, a paired Student’s T-test was used to compare values of weight, age, height and BMI of both groups and to determine homogeneity between the two groups. Concerning sway and CoP data, we used a MANOVA for all the variables of interest. A comparison Pre Caffeine/energy drink between groups (CoP and Sway) during all task was performed to ensure similarities between groups. Subsequently, a comparison between groups (MANOVA) was performed post Caffeine/energy drink consumption to assess the role of both substances. The test results were adjusted using a Bonferroni correction to identify the different parameters with a significant difference. In this study, a P value of 0.05 or lower was considered statistically significant.

A Paired Student T-test ( Table 2) was used to compare values of weight, age, height and BMI of both groups. None of the variables showed statistically significant results, evidencing homogeneity in the characteristics of subjects between the groups.

In the comparison of the caffeine group ( Table 3), a significant increase (p ≤0.05) was found only in the AP sway in the eyes closed test while standing on a stable surface. The AP sways before caffeine ingestion was 1.65 ± 0.41, and 30 minutes after the ingestion it was 2.85 ± 1.1. For CoP in any of the BALT’s, including the EO test, the results were statistically significant.

When comparing pre and post average of AP sway, ML sway and COP in the energy drinks group ( Table 4), no statistically significant results were found in any of the variables measured in any of the BALT’s.

No statistically significant results were found for the comparison of the average of AP sway, ML sway, and COP of energy drinks and caffeine post BALT’s ( Table 5).

In a secondary analysis, a comparison of the data over time (0, 15, 30 seconds) of each balance test was performed. In this analysis, we also observed a tendency of the energy drinks to alter the postural control, more significantly in the middle of the test and before subjects were able to recover overall postural control at the end of the test.

First, we assessed postural stability (pre and post average) and compared the effects of caffeine and energy drinks in the individual groups to determine the effects of consumption individually during eight sensory conditions.

In the caffeine pre and post comparison, 5/8 of the BALT's results showed an increase in the average of CoP. However, none of the results were statistically significant. A statistically significant difference (P≤0.05) was found only in AP sways values in the EC test in the stable surface. Nevertheless, the average of CoP in the same test did not show statistically significant difference suggesting that overall postural control was maintained. Even though in the present study the consumption of caffeine alone did not have a significant effect in altering the postural control, available evidence is inconsistent with this conclusion. In their study, Kim et al. ²⁴ measured postural control during EO, EC and a changed base of support after an approximate intake of 73 mg of caffeine. They concluded that after 40 minutes of caffeine intake, there was not a significant alteration to postural control in the healthy subjects. Compared with our study, they gave an amount of caffeine which was significantly less than the caffeine administrated in our study (200mg); but overall results were similar. Meanwhile, following the present study, Mcnerney et al. ²⁵ measured postural control after altering the system's associate to postural control after the ingestion of 300mg of caffeine and found a significant difference in the eyes closed, platform sway-referenced test. However, authors made a general conclusion that caffeine did not produce a clinically significant effect in healthy young participants. Furthermore, Franks et al. ²⁶ measured body sway of young subjects during stance with EO and EC after ingesting 300mg of caffeine per 70 kg of weight. The researchers observed a significant increase in body sway (eyes open) after 40 minutes, but subjects recovered stability at later times (100, 160 minutes). A similar behavior was observed with the eyes closed test of this study; but it was not significant, evidencing a tendency of the subjects to improve postural stability with time. It is important to note that in Franks et al. study, subjects’ weight was considerate to determine the amount of caffeine administrated, which should be taken into consideration for the comparison of the results of our study, and for the design of future studies assessing the effect of caffeine in postural control.

In the energy drinks pre and post comparison, 6/8 of the BALT's showed an increased average of COP, 5/8 BALT’s in the AP sway and 6/8 BALT’s in the ML sway 30 minutes after the consumption of the energy drink. The previous results suggest a negative effect on the postural control mechanism due to a possible stimulatory effect of the energy drinks in the CNS. The BALT’s EO, EC, EO HUD, EC HUD, EC MAT were affected in the three measurements (CoP, AP sway, ML sway), while EC HUD MAT was only affected in the CoP, and EO HUD MAT in the ML sway only. EO MAT was the only BALT’s not altered in any of the three measurements. Even though we observed a tendency of an increased postural instability after the energy drinks consumption, none of the results were statistically significant. No association between the systems altered, and the results could be established since there was not a pattern of the altered/canceled system and the increased instability observed in the BALT's. Enriquez et al. ¹⁷ also measured body sway with the eyes closed and eyes open only 1 hour after the ingestion of energy drinks (160 mg of caffeine). In the study, 23 young subjects presented an increased sway in both conditions, but none of the results were statistically significant which correlates with our findings. Available evidence at the moment seems to indicate that energy drinks appear to make small increases in body sway. However, it does not impair the postural control mechanism significantly, at least in healthy subjects with a dose of 160 mg of caffeine given through energy drinks. How the postural control mechanism responds to an even higher dose of energy drinks or in other populations at risk for falls such as older adults remains unknown, however a stepping stone for future studies.

Secondly, we compared the effects of caffeine versus energy drinks, to determine any differences in post test results regarding postural instability between the subjects of both groups, even though there was a difference of 40 mg of caffeine between the energy drink (160 mg) and caffeine group (200 mg). The average of CoP, AP sway and ML sway in the caffeine group seems to be more affected in the BALT's performed on the unstable surface (foam mat), while the energy drinks showed more instability in the stable surface. Albeit, an association or correlation between the system altered/canceled (specific BALT’s) and the effect between both groups could not be established, as the results were not consistent. Although, the energy drinks, in addition to the 160 mg of caffeine, contain potential stimulating ingredients. However, the difference of 40 mg of caffeine between groups was not a major factor as none of the post test results (CoP, AP sway, and ML sway) were statistically significant in the comparison between subjects of both groups. The results of this comparison show that the sensory re-weighting system of the twenty healthy young subjects in both groups was able to adjust and re-adjust the sensory inputs appropriately to maintain overall postural control despite the postural perturbations during the BALT’s and the stimulatory effect of the intake of the CNS stimulants caffeine and energy drinks.