Neuromuscular Adaptations after Blood Flow Restriction Training Combined with Nutritional Supplementation : A Preliminary Study

Blood fl ow restriction training (BFRT) has been shown to be an eff ective alternative technique to conventional resistance training to increase skeletal muscle hypertrophy and strength. However, neuromuscular response to BFRT in combination with nutritional supplementation has rarely been studied. Th e purpose of this study was to investigate the eff ects of BFRT combined with creatine monohydrate (CrM) and/or hydroxymethyl butyrate (HMB) on skeletal muscle size and strength. Fift een healthy males were randomly divided into three groups: a) BFRT without supplementation (C-BFR); b) BFRT with CrM supplementation (0.3 g / kg-1) (CrM-BFR); and c) BFRT with CrM (0.3 g / kg-1) and HMB (3 g) supplementation (CrM + HMB-BFR). Participants performed elbow fl exion exercise (30% of maximal isometric voluntary contraction (MIVC)) at 30% of total occlusion pressure, twice a week, for three weeks. MIVC of the elbow fl exion and brachial biceps muscle thickness were assessed preand post-training. Th ere was no signifi cant group-by-time interaction for MIVC values (p>0.05). Biceps muscle thickness was signifi cantly increased from preto post-test in all groups (p<0.05). Th e C-BFR group obtained a greater value of eff ect size (d=2.2). Th ese fi ndings suggest that 3 weeks of CrM and HMB supplementation had no infl uence on BFRT-induced neuromuscular adaptations.


Introduction
Resistance training (RT) is essential to promoting neuromuscular adaptations in a large range of the population.Consequently, the American College of Sports Medicine (ACSM) guidelines recommend using loads of ≥70% of one repetition maximum (1RM) to achieve muscular hypertrophy and strength gains (American College of Sports Medicine, 2009).While these guidelines are appropriate for healthy individuals, inactive adults and clinical populations may have signifi cant limitations to using the load of RT recommended for ACSM; therefore, other options are necessary to improve musculoskeletal fi tness (Chulvi-Medrano, 2011).For these populations, low-load resistance training (20%-30% 1RM) with blood fl ow restriction (LL-BFR) has been deemed an alternative technique to conventional RT (Chulvi-Medrano, 2011;Jeremy Paul Loenneke & Pujol, 2009;Pope, Willardson, & Schoenfeld, 2013;Slysz, Stultz, & Burr, 2016).Th is training technique, also called Kaatsu® training, has been shown to be an eff ective method for increasing muscle size and strength in clinical settings and athletic populations (Amani, Sadeghi, & Afsharnezhad, 2018;Martín-Hernández, Marín, 2011;Martín-Hernández, 2011;Meester, Stodden, Brian, True, & Cardon, 2016).During LL-BFR exercise, partial blood fl ow restriction of the exercised muscle is achieved by proximal compression of the limb.LL-BFR training usually consists of 2-3 sessions per week, 3-5 sets of 15 repetitions or sets carried out to volition-al failure, and 30-60-second rest intervals between sets (Martín-Hernández, 2011).
Nutritional supplementation combined with RT has been shown to yield signifi cant increases in muscle size, strength, and power in greater magnitude than RT alone (Buford et al., 2007;Helms, Aragon, & Fitschen, 2014;Pearson, Hamby, Russel, & Harris, 1999).Specifi cally, creatine monohydrate (CrM) and hydroxymethyl butyrate (HMB) supplementation combined with RT have been shown to be eff ective for inducing neuromuscular adaptations in trained and untrained individuals (Buford et al., 2007;Helms et al., 2014;Portal, Eliakim, Nemet, Halevy, & Zadik, 2010;Wilson et al., 2013).Oral intake of CrM increases both intramuscular creatine and phosphocreatine concentrations leading to a concomitant increase in body mass and performance (Balsom, Söderlund, & Ekblom, 1994;Birch, Noble, & Greenhaff , 1994;Helms et al., 2014).Th ese ergogenic eff ects may be caused by the availability of the amino acid leucine and some derivative metabolites, since the β-isocaproate inhibits proteolysis (Nair, Schwartz, & Welle, 1992;Nissen et al., 1996).Th e positive eff ects of RT with HMB supplementation are also consistent.Jówko et al. (2001) demonstrated in untrained individuals that the increases skeletal muscle mass and strength following RT with HMB were superior in comparison to those obtained with RT alone aft er 3 weeks of training.In addition, Wilson et al. (2014) found that this strategy was eff ective to enhance RT training-induced gains in strength, power, and muscle mass in resistance-trained males.
While there is a growing body of evidence highlighting the potential benefi ts of LL-BFR exercise on skeletal muscle hypertrophy and strength, the eff ects of this strategy combined with nutritional supplementation on neuromuscular adaptations responses remain unknown.
Th erefore, this study aimed to investigate the eff ects of an LL-BFR exercise programme with and without CrM and HMB supplementation on skeletal muscle hypertrophy and strength adaptations.We hypothesized that the LL-BFR-induced hypertrophy and strength increases combined with CrM and HMB would overcome those obtained with LL-BFR alone, since the CrM and HMB supplementation combined with conventional RT has shown a synergistic eff ect (Jówko et al., 2001).

Participants
Fift een healthy male untrained college students (age: 23.3 ± 2.6 years; body mass: 78.1 ± 12.68 kg; height 1.78 ± 0.07 m) were recruited to participate in this study.Th e participants were randomly allocated to one of the three groups: a) blood fl ow restriction without supplementation (C-BFR, n=5), b) blood fl ow restriction with creatine monohydrate supplementation (CrM-BFR, n=5), and c) blood fl ow restriction with creatine monohydrate plus hydroxymethyl butyrate supplementation (CrM + HMB-BFR, n=5).Th e criteria for inclusion in the study were: resistance-trained men who were 20-30 years old with at least 6 months of experience in RT.Participants with cardiovascular risk, food intolerances, or specifi c allergies to the supplements, neuromuscular disorders or any acute or chronic disease were excluded from the study.Likewise, all participants should not have used nutritional supplementation or drugs within six months prior to the commencement of the study.Th e study was approved by the Ethics Committee of the University of Valencia (procedure number H1419281092018) and conducted accordingly with the Declaration of Helsinki.Following the explanation about the risks and benefi ts of this study, participants signed their written informed consent.

Procedures
Anthropometric measurements (body mass, height, body mass index), maximal isometric voluntary contraction (MIVC), biceps muscle thickness (BMT), and total occlusion pressure (TOP) were obtained prior the study.Aft er two familiarization sessions of testing and protocols, participants were randomly divided into three training groups: a) blood fl ow restriction without supplementation (C-BFR), b) blood fl ow restriction with creatine monohydrate supplementation (CrM-BFR), and c) blood fl ow restriction with creatine monohydrate plus hydroxymethyl butyrate supplementation (CrM + HMB-BFR).All participants performed the biceps curl exercise at 30% of MIVC and 30% of TOP, 3 sets of 15 repetitions, twice per week, for three weeks.Elbow fl exion strength and biceps muscle thickness were assessed before and aft er training.

Participant's characteristics and anthropometric measurements
Body mass (kg) was measured with the participants in a standing position on electrical bioimpedance equipment (Omrom -HbF-510W), and height (m) was measured with each participant at the same position on a stadiometer (Seca® 217).

Maximal isometric voluntary contraction (MIVC).
Isometric strength was evaluated through MIVC from the dominant arm of each participant using a load cell (Mutonic® SP51 "HiLine" V6.10).Th e participants were placed in a standing position, and their elbow positioned at 90º of fl exion and monitored with an analogic goniometer.Participants performed two attempts of maximal voluntary isometric contractions for fi ve seconds, with rest periods of 180s between them.In short, participants were instructed to perform an elbow fl exion action as forcefully as possible during contraction time.Th e highest value of two attempts was used for statistical analysis.

Bicep muscle thickness.
Bicep muscle thickness was recorded using an ultrasonographic technique (Sonosite M-Turbo).Th e measurements were performed with the participants in a supine position with their arms extended and relaxed.Before measurements, the participants rested for 15 min to allow fl uid shift s to occur and all muscle thickness measurements were performed by an experienced physiotherapist.Biceps muscle thickness values were measured by placing the probe perpendicular without depressing the skin at the specifi c landmark that was identifi ed at 60% of the distance from the acromion process of the scapula to the lateral epicondyle of the humerus.

Total occlusion pressure (TOP)
. Th e value of TOP was individually registered through Doppler ultrasound.For this measure, a pressure cuff (57 cm length × 9 cm width; Riester Komprimeter, Riester, Jungingen, Germany) was attached to participant's axillar region and then progressively infl ated, while the arterial blood fl ow was monitored using Doppler ultrasound.When brachial arterial blood fl ow was interrupted, the arterial occlusion pressure level was recorded (100% of TOP).For all BFR protocols, 30% of TOP was used.
Training protocols.Prior to starting the training protocols, participants performed two familiarization sessions with LL-BFR exercise at 20% of MIVC and 20% of TOP.A 48h interval was allowed between familiarization sessions and testing before starting the training programme.For training protocols, all participants performed the biceps curl exercise at 30% of MIVC and 30% of TOP individual with the dominant arm.In each training session, the participants performed 3 sets of 15 repetitions with a 60-second interval between sets and a contraction cycle duration of 2 seconds in the concentric phase and 2 seconds in the eccentric phase.Th e pressure restriction was maintained throughout the exercise and rest intervals.Th e training programme was carried out with a frequency of 2 days per week (Monday and Th ursday), for 3 weeks.All training sessions were supervised by a personal trainer.

Nutritional supplementation.
Prior to the study, participants recorded their dietary pattern for a week, and a nutritionist instructed the participants to maintain their normal diet based on the data collected in the study.Besides normal dietary, the participants from the CrM-BFR group was supplemented with creatine monohydrate (rate of 0.3 g/kg body mass), whereas the participants from CrM + HMB-BFR group received the same amount of creatine monohydrate of the CrM-BFR group combined with 3g of supplementation of hydroxymethyl butyrate (Scitec Nutrition®).Th e C-BFR group received no supplementation.Nutritional supplementation doses used in this study were based on the fi ndings of previous studies (Buford et al., 2007;Wilson et al., 2013).Nutritional supplementation was administered 10 minutes before beginning each training session in a double-blind fashion.

Statistical analysis
Th e results are presented as mean and standard deviations (SD).Th e comparison between paired samples (intragroup) was carried out using a t-test to compare maximal isometric voluntary contraction and biceps muscle thickness in each group before and aft er the training programme.Adjustment for multiple comparisons was made with Bonferroni's correction.Th e analysis of variance (two-factor ANOVA) was also performed followed by post hoc DMS multiple comparisons to make intergroup comparisons.Th e level of signifi cance was set at p<0.05.SPSS 18.0 soft ware, licensed from the University of Alicante, was used for statistical analysis.Th e eff ect size was calculated using the Cohen test ([post-test mean -pre-test mean] / pre-test standard deviation).To determine the eff ect size of the intervention, applied the values for trained subjects were applied (Rhea, 2004): trivial eff ect d<0.25; small eff ect d= 0.25 -0.50; moderate eff ect d= 0.50-1.0;large eff ect d>1.0.Finally, the percentage increase in each group was calculated using the following formula: (post-intervention mean -pre-intervention mean) / pre-intervention mean × 100.

Maximal isometric voluntary contraction
Th ere was no time eff ect × interaction for MIVC for all groups from pre to post-test (p>0.05).MIVC was increased in 11.20%, 31.60%, and 3.66% in the C-BFR, CrM-BFR and CrM + HMB-BFR groups, respectively, from pre-to post-training (Table 1).No signifi cant increase was found between groups at pre-test and posttest (p>0.05).

Biceps muscle thickness
Th ere was a signifi cant main eff ect of time and group interaction for biceps muscle thickness (p<0.05).Biceps muscle thickness was increased in 12.5%, 8.88%, and 13.12% in the C-BFR, CrM, and CrM + HMB-BFR groups respectively, from pre-to post-training (Table 2).Th ere was a signifi cant diff erence between the C-BFR group and CrM + HMB-BFR group at post-training (p<0.05).

Discussion
Th e main fi ndings of this investigation were: a) Isometric strength was not infl uenced by blood fl ow restriction training or nutritional supplementation; b) Low-load RT with blood fl ow restriction induced increases in biceps muscle hypertrophy regardless of nutritional supplementation.

Maximal isometric strength
Despite the fact that other studies have demonstrated an advantage in strength gains in applying low-load resistance training with blood fl ow restriction compared to the same protocol without BFR (Slysz et al., 2016) and a similarity to high-load resistance training (Laurentino et al., 2012;Takarada et al., 2000), in our study we did not observe changes in isometric peak torque of elbow fl exors following 3 weeks of BFRT.Similar fi ndings were reported in a recent study in which the isometric peak torque of the knee extensors was not signifi cantly changed following 6 weeks BFRT (Cook, Scott, Hayes, & Murphy, 2018).In addition, in this study, a reduction of 2% in the central activation was observed.Th ese results suggest that the involvement of the central nervous system following BFR resistance exercise as strength improvements may not be of neural origin.
In contrast, in Cook's study, a signifi cant increase of 13% in leg extension 1RM was observed.Although we did not evaluate elbow fl exors 1RM in our study, it has been suggested that nonspecifi c strength assessment, such as in isometric of isokinetic testing, may more precisely refl ect the response to diff erent training protocols (Buckner et al., 2017).Regarding the overall strength observed in the present study, we can speculate that the training mode through of isotonic contractions and the lack of signifi cant change in isometric muscle strength may mask neural adaptations assessed through an isometric contraction.Altogether, the low interference of LL-BFR on neural adaptation and testing specifi city could have infl uenced the response in strength gains aft er the training period.However, short-term training (3 weeks) perhaps would have been insuffi cient to reach signifi cant changes in strength following LL-BFR.

Muscle hypertrophy and nutritional supplementation
Regarding increases in muscle mass, our results showed that BFR protocols were effi cient to increase muscle hypertrophy, regardless of nutritional supplementation.Th ese data are consistent, since a variety of studies has pointed out higher muscle mass gain in LL-BFR in comparison to LL without BFR (Slysz et al., 2016) and similar eff ects to high-load resistance exercise (Laurentino et al., 2012;Martín-Hernández et al., 2013).However, no study to our knowledge has investigated the eff ect of nutritional supplementation combined with LL-BFR on muscle size.Th e Position Stand of the International Society of Sports Nutrition has demonstrated the ergogenic eff ects of supplementation of CrM combined with RT (Buford et al., 2007).For instance, Souza-Junior et al. ( 2011) reported increases in maximal strength, isokinetic peak torque and muscle mass aft er 8 weeks of RT programme (8-10 RM) in bench press and back squat exercises associated with the supplementation of 20 g of CrM.In another study, Antonio and Ciccone (2013) demonstrated that 5g of creatine monohydrate ingestion post-exercise, twice a week, induced increase in fat-free mass and loss of fat mass following a 4-week periodized-resistance training programme (3 sets × 5-10 reps).
Previous research has evaluated the eff ectiveness of CrM combined with HMB supplementation on muscle mass and strength gains, and the results are confl icting (Jówko et al., 2001;O'Connor & Crowe, 2007)  Lastly, the discrepancies between our fi ndings compared to the previous studies regarding nutritional supplementation combined with LL-BFR may be, at least partially, explained by the diff erences in the dose of supplementation, the duration of the study, and the load used in LL-BFR protocols.
In conclusion, three weeks of LL-BFR induced increases in muscle hypertrophy but not on strength gains.In addition, CrM and CrM plus HMB supplementation showed no additional eff ect of LL-BFR-induced neuromuscular adaptations.
Th e current study reveals some potential limitations.First, this is a preliminary study, and the small sample size limits statistical power.Second, the lack of dietary monitoring using a normalized isocaloric diet also could have aff ected our results.Th ird, an LI-BFR with HMB supplementation and placebo groups could be included in study design.Lastly, the duration of this study could be longer (more than 4-6 weeks).
BLOOD FLOW RESTRICTION TRAINING | I. CHULVI-MEDRANO ET AL.

TABLE 1
Maximal Isometric Voluntary Contraction for C-BFR, CrM-BFR, and CrM + HMB-BFR Groups from Pre-to Post-test

TABLE 2
Outcomes for the Biceps Muscle Thickness Before (pre-test) And After (post-test) Intervention Signifi cant diff erence from pre-to post-training (p<0.05);†Signifi cant diff erence between C-BFR group and CrM + HMB-BFR group at post-training (p<0.05).BLOOD FLOW RESTRICTION TRAINING | I. CHULVI-MEDRANO ET AL.