Twelve healthy older male and female volunteers (63±3 y, 7 men: 5 women, BMI: 29±1 kg/m ²) with no previous chronic disease history were recruited via an advertisement on a local radio station. Before enrolment into the study, all volunteers had an assessment of previous medical history and an ECG to check for any subclinical arrhythmias. The ECG’s were approved by a clinically qualified physician. All volunteers provided written informed consent prior to the start of this study. This study was approved by The University of Nottingham Medical School Ethics Committee (B28102015 SoMS GEM BBC) and conformed to The Declaration of Helsinki.

The study involved two visits by the study participants, before and after the 4-week HBET ( Figure 1), to the University of Nottingham Medical School, at the Royal Derby Hospital. All assessments at these visits were conducted by researchers from the MRC-ARUK Centre for Musculoskeletal Ageing Research. At each visit, volunteers provided a baseline urine sample for the muscle mass assessment by D ₃-Creatine before undergoing measures of height, weight (Marsden Weighing Group, UK), resting heart rate and blood pressure (Omron M5-1 digital BP monitor, Omron, UK). Each volunteer had the thickness, fascicle length ( L _f ) and pennation angle ( θ _pen ) of their vastus lateralis muscle , and the cross-sectional area (CSA) of their quadriceps measured by ultrasound (Mylab 70, Esaote Biomedica, Genova, Italy), using the protocol described by Franchi et al. (2014). Volunteers then completed a number of muscle function assessments:

MVC was measured at two knee angles (60 and 90°), whilst isokinetic (eccentric/concentric) contractions were measured over a 70° range (20–90°) at three different speeds (60, 180 and 240°/sec). For both measures, horizontal seated leg extension was set at 0°. Following the muscle function assessments, volunteers ingested 30mg of D ₃-Creatine dissolved in 100ml H ₂O for muscle mass assessment, before providing a pooled 24h urine collection and single urine samples at 48 and 72 h post consumption ( Clark et al., 2014).

Volunteers were required to complete 3 sets of 12 repetitions across 8 different exercises, every day, for 4 weeks. Each exercise was designed to be easily incorporated into activities of daily living to minimise the time-commitment required (e.g. bicep curls whilst cooking, see Figure 1). In total the exercise programme consisted of: squats, lunges, calf raises, bicep curls, triceps extensions, semi-incline press-ups, oblique twists and deadlifts. Participants were instructed to take 2 seconds to perform each phase (eccentric and concentric) of each exercise and to hold for 2 seconds at the mid-point of each exercise. Progression via increased resistance or time-under-tension was encouraged once the full complement of repetitions could be completed. Compliance to exercise was measured by self-report (see self-report log in Figure 1). An exercise would only be marked as complete if 3 sets of 12 repetitions had been performed within a day.

Using a creatine-tracer measure of muscle mass ( Clark et al., 2014), D ₃-creatine and D ₃-creatinine enrichment, plus unlabelled creatine and creatinine in the urine was measured using high-performance liquid chromatography/mass spectrometry (HPLC/MS), as detailed in ( Stimpson et al., 2012). Total muscle mass was calculated using the following equation:

 Total muscle mass = ( M W U n l a b e l l e d M W l a b e l l e d ) × ( A m o u n t   o f   D 3 − C r   d o s e d ( g ) − A m o u n t   o f   D 3 − C r   e x c r e t e d   ( g ) ) ( m e a n   s t e a d y − s t a t e   D 3 − C r e a t i n e   e n r i c h m e n t   r a t i o ÷ 4.3 ( g / k g )

                                                                                                                                         ( Clark et al., 2014),

where MW _Unlabelled and MW _labelled represents the molecular weights of both unlabelled and labelled creatine, respectively. The estimated creatine pool size is then divided by 4.3 g/kg, which reflects the concentration of creatine in wet muscle.

Descriptive statistics were carried out on all datasets and checked for a normal distribution using the Shapiro-Wilk normality test. Datasets were analysed using paired Student’s t test to assess the effect of HBET, with GraphPad Prism Software v5 (La Jolla, CA, USA). All data is presented as mean ±SEM. Significance was set at P<0.05.

Using data from the self-report training logs, overall compliance to the HBET was 87.3±3.0% over the 4-week period of training. Adherence to each individual exercise was >86%, with the calf-raises being the most completed exercise (91%). No significant changes were found following HBET for body weight (79.3±4.7 vs. 78.9±4.6 kg), resting heart rate (71.9±3.1 vs. 71.4±3.9 bpm) or blood pressure (systolic: 144.4±3.4 vs. 140.7±4.2, diastolic: 92.2±2.5 vs. 89.0±2.4; average of 3 measurements).

HBET did not elicit significant changes in leg extension 1-RM, handgrip strength or SPPBT; there was however, a trend for an increase in leg extension 1-RM (45.7±5.9 vs. 49.6±6.0 kg, P=0.08; Figure 2A). HBET did elicit significant increases in leg power (276.7±38.5 vs. 323.4±43.4 W, P<0.05; Figure 2B) and MVC at 60° (154.2±18.4 vs. 168.8±15.2 Nm, P<0.05; Figure 2C) and 90° (152.1±10.5 vs. 159.1±11.4 Nm, P<0.05; Figure 2D). There were also significant increases in peak torque recorded at 60°/sec during both seated flexion (74.7±8.3 vs. 80.8±8.6 Nm, P<0.05; Figure 2E) and extension (114.9±10.2 vs. 123.8±9.4 Nm, P<0.01; Figure 2F) after HBET.

CSA of the thigh at 50% of thigh length significantly increased (57.5±5.4 vs. 59.0±5.3 cm ², P<0.05; Figure 3A) after HBET. However, there was no significant increase in muscle thickness, fascicle length ( L _f ) or pennation angle ( θ _pen ) of the m. vastus lateralis. There was a trend for an increase in muscle thickness (25.0±1.6 vs. 25.5±1.7 mm, P=0.08; Figure 3B) after HBET.

Corresponding with no significant change in body weight, there was no significant difference in whole-body muscle mass as estimated by D ₃-Creatine ( Figure 3C) after HBET (23.9±2.1 vs. 22.8±2.2 kg).