Subjects
Fifteen young men (26 ± 2 years) participated in this study. All subjects were healthy and recreationally active (2–3 h physical activity per week). Inclusion criteria were: age between 18 and 35 years, experience in resistance training (> 1 year), non-smoker, no neurological, acute orthopedic injuries as well as chronic diseases and no history of deep vein thrombosis. Additionally, participants with acute lower extremity injuries and uncontrolled hypertension were excluded from the study.
Approval of the study was obtained by the local ethics committee and all procedures were in accordance with the latest revision of the Declaration of Helsinki. All participants were informed about the potential risks before written informed consent was given.
Study design
To investigate the effects of BFR on muscle excitation, metabolic accumulation, muscle cell swelling and recovery, a repeated measures cross-over design was implemented. One week before start of the study, participants underwent a preliminary screening which included a medical anamnesis as well as an examination to confirm the compliance with the abovementioned inclusion criteria. After eligibility, all fifteen participants completed two isometric resistance exercise sessions of the knee extensors on two different days in a random and counterbalanced order: 1) low-load (20% MVC) resistance exercise under normal blood flow conditions and 2) low-load (20% MVC) resistance exercise with simultaneous BFR. Prior to measurements, all participants were instructed to avoid strenuous and unaccustomed exercise for 72 h and to follow a 12 h fasting period. All sessions were performed at the same time of the day (between 8 a.m. and 12 a.m.). To ensure an adequate wash-out and recovery period, both sessions were separated by a minimum of 7 and a maximum of 10 days. All outcome assessors were blinded to the respective exercise session.
Procedures
Exercise protocols
Low-load resistance exercise (LL)
During this session, low-load resistance exercise was performed at 20% of each individual’s maximum voluntary torque. The subjects were sitting in front of a computer screen watching a red line representing the torque produced by the subjects. This line had to be matched with a black line representing the individual 20% MVC torque. The subjects completed three sets of 90 s of isometric knee extension (knee angle 90°). A resting period of 30 s was allowed between each set. A rest period of 30s was chosen since findings from a previous meta-analysis have demonstrated that lower resting periods seem to augment certain muscular adaptations (e.g. muscle strength) compared to longer resting periods in LL-BFR training [27].
Low-load resistance exercise with blood flow restriction (LL-BFR)
During this condition, participants performed the same exercise but had a 12 cm pneumatic nylon cuff [Tourniquet Touch TT20, VBM Medizintechnik GmbH, Germany] applied around the most proximal portion of both thighs. Before starting the session, arterial occlusion pressure (AOP) was determined in a sitting position for each participant. The cuff pressure was steadily increased until the arterial pulse at the posterior tibial artery was no longer detected by Doppler ultrasound [Handydop, Kranzbühler, Solingen, Germany]. This point was defined as 100% of arterial occlusion. During exercise, cuff pressure was preset to 50% of each individual’s AOP and kept inflated during the entire session including the 30 s interset rest periods. This pressure was chosen to be able to compare with previous studies being conducted under dynamic contraction modes [10, 28].
Dependent variables
Muscle activation
Before the electrodes were attached to the vastus lateralis (VL), the skin of the subjects was shaved and cleaned with disinfectant. Electromyographic muscle activation (EMG) was assessed using biopolar surface electrodes [Blue sensor P, Ambu, Bad Nauheim, Germany] at the VL at 50% of femur length (from greater trochanter to the inferior border of the lateral epicondyle) and 2 cm interelectrode distance. The axis of both electrodes was aligned with the muscle fiber orientation. A reference electrode was placed on the patella, and all signals were pre-amplified (1000 ×), band-pass filtered (10–1000 Hz) and sampled at 2048 Hz using a TMSi refa system (TMSi, Twente, The Netherlands). Additionally, electrode positions were marked with a surgical pen in order to replicate the exact locations during the subsequent session. Maximal muscle activity was obtained during the initial three MVCs and the highest rectified EMG value obtained in the MVCs was taken as the maximal EMG activation used for normalizing the EMG data during the subsequent 90s intervals. The VL activation during the 90s intervals was determined by calculating the root mean square (RMS) of the rectified EMG of 10 s intervals.
Metabolic accumulation
Metabolic accumulation was estimated via lactate concentration levels. Before the exercise session, following each of the three sets as well as 15 min post completion, 20 μl of capillary blood were obtained from the ear lobe. All samples were analyzed via enzymatic-amperometric methods using a Biosen S-Line lactate analyzer from EKF Diagnostics [Cardiff, UK].
Muscle cell swelling
Muscle cell swelling was calculated by measuring acute changes in muscle thickness using b-mode ultrasound. Muscle thickness of the rectus femoris (RF) muscle was measured at 50% of femur length before the exercise session, immediately after each set as well as following 15 min after completion. An ultrasound device [8 MHz, ArtUs EXT-1H; Telemed, Vilnius, Lithuania] with a 60 mm linear transducer was used to acquire sagittal images at the mid distance between the medial-lateral boarders of RF. During the measurement, participants were positioned in a sitting position with their knee and hip angles at 90°. The baseline picture was acquired after a resting period of 20 min, which was implemented to allow fluid shifts. During the whole procedure, participants were instructed to relax their muscle as much as possible. Additionally, a sufficient amount of ultrasound gel was used in order avoid pressure to the skin causing muscle compression. This was ensured by confirming a clearly visible ultrasound gel layer on each image. During each time point, three images were obtained.
For offline analyses, the shortest distance between upper and lower aponeurosis was measured at 25, 50 and 75% of each image. Each image was analyzed three times and the mean of all distances and images was used for further statistical analyses. Reliability of the ultrasound image analyses was confirmed by a very low coefficient of variation of 1.46%.
Decrements in maximum voluntary torque
Unilateral, isometric maximum voluntary contraction (MVC) torque at 90° knee extension was measured before, immediately after as well as 15 min following completion of the exercise session using an isokinetic dynamometer [ISOMED 2000, Ferstl, Germany]. Subjects were placed in supine position with restricted shoulders and hips. During the entire procedure knee and hips were fully extended. In total, three trials were conducted with a rest period of 1 min. The mean of all three trials was used for data analysis. All data were normalized to body weight.
Perceptual responses
To measure the rating of perceived exertion (RPE), a conventional BORG scale [29] was utilized. Participants were instructed to rate their current RPE on a rating scale reaching from 6 ‘very, very light’ to 20 ‘very, very hard’. RPE was rated before the exercise session, following each of the three sets as well as 15 min after completion.
Statistics
Normal distribution and homogeneity of variances was checked for all variables. To test for difference in the EMG during the MVCs between the sets 1–3, a repeated measures ANOVA (rmANOVA) was calculated with the factors time (Pre, Post 15) and condition (LL-BFR, LL). Differences in EMG between the sets were tested by a rmANOVA with factors time (Set 1, Set 2, Set 3) and condition (LL-BFR, LL). For changes in MVC torque, a rmANOVA with factors time (Pre, Set 3, Post 15) and condition (LL-BFR, LL) was conducted.
For all other variables, individual rmANOVAs with factors time (Pre, Set 1, Set 2, Set 3, Post 15) and condition (LL-BFR, LL) were calculated. This included EMG during the 90s intervals, muscle thickness, lactate concentrations and RPE. In case of significant interactions effects, an analysis of simple effects was conducted.
Software package SPSS 24.0 [IBM, Armonk, USA] was used for all statistical analyses. Data is presented as mean ± standard deviation, if not indicated otherwise. The level of significance was set to p < 0.05 for all tests.