Scientists have attempted to increase training-induced adaptations through a number of protocols, which generally aim at augmenting and/or speeding skeletal muscle regeneration(Reference Renault, Piron-Hamelin and Forestier1–Reference Ge, Wu and Warnes3). Effective training protocols for increasing lean body mass and strength often leave athletes in a fatigued state, which can limit overall training frequency(Reference Carpinelli and Otto4). Oral administration of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) has been associated with increases in lean body mass(Reference Gallagher, Carrithers and Godard5, Reference Jowko, Ostaszewski and Jank6), strength(Reference Nissen, Sharp and Ray7) and power(Reference Kraemer, Hatfield and Volek8). HMB is presently thought to work by speeding regenerative capacity as evidenced by increased protein synthesis and decreased muscle protein breakdown, damage and soreness following high-intensity exercise(Reference Nissen, Sharp and Ray7). Recent data suggest that HMB acts through multiple mechanisms that include the simultaneous stimulation of the mammalian target of rapamycin, the major kinase directing translation initiation of muscle protein synthesis(Reference Norton and Layman9, Reference Baxter, Carlos and Thurmond10), and the inhibition of the ubiquitin–proteasome(Reference Smith, Mukerji and Tisdale11, Reference Smith, Wyke and Tisdale12) pathway, the major regulatory system of protein degradation in skeletal muscle(Reference Mitch and Goldberg13).
The overwhelming majority of studies using HMB have examined its results after one or more weeks of training and supplementation(Reference Gallagher, Carrithers and Godard5–Reference Nissen, Sharp and Ray7, Reference Panton, Rathmacher and Baier14, Reference Neighbors, Ransone and Jacobson15). To date, only one acute HMB study has been conducted. In this study, Wilson et al. (Reference Wilson, Kim and Lee16) examined the acute effects of an oral Ca HMB (HMB-Ca) supplement on sixteen untrained males using a unilateral, isokinetic leg extension-based training protocol. These researchers found that HMB-Ca consumed before exercise blunted the rise in lactate dehydrogenase relative to the supplement consumed following exercise or a placebo. This study was conducted using an untrained population and controversy exists over whether HMB is effective in highly trained individuals(Reference Wilson, Wilson and Manninen17). In addition, the training stimulus used in that study was neither traditional nor practical for eliciting desirable body mass and strength changes in a trained population. Finally, HMB-Ca used by Wilson et al. (Reference Wilson, Kim and Lee16, Reference Wilson, Wilson and Manninen17) has a slow rate of appearance, taking approximately 60–120 min to reach peak plasma values(Reference Fuller, Sharp and Angus18). Due to the slow rate of appearance of HMB-Ca and the need for its ingestion much before training, HMB-Ca may be impractical for everyday use in an exercise regimen.
Recently, Fuller et al. (Reference Fuller, Sharp and Angus18) observed that an HMB free acid (HMB-FA) supplement was absorbed more rapidly, peaked faster (30 min) and had a greater clearance to tissues than an HMB-Ca supplement, thus shortening the length of time needed between supplementation and exercise.
Research is lacking on HMB and resistance-trained athletes using a practical and high-intensity training stimulus. Therefore, the purpose of the present study was to investigate the acute effects of an HMB-FA supplement administered just before a bout of resistance exercise on serum indices of muscle damage, inflammation, perceived recovery, muscle protein breakdown, and anabolic and catabolic hormone status in resistance-trained men.
Methods
Subjects
A total of twenty resistance-trained males aged 21·6 (sem 0·5) years with an average squat, bench press and dead lift of 1·7 (sem 0·04), 1·3 (sem 0·04) and 2·0 (sem 0·05) times their body weight were recruited for the study (Table 1). Subjects could not participate if they were currently taking anti-inflammatory agents, any other performance-enhancing supplement or if they smoked. The present study was registered at ClinicalTrials.gov (registration no. NCT01508338). The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and the University of Tampa Institutional Review Board approved all procedures involving human subjects. Written informed consent was obtained from all subjects.
Table 1 Subject characteristics (Mean values with their standard errors, n 20)
HMB, β-hydroxy-β-methylbutyrate; DEXA, dual-energy X-ray absorptiometry; BF, body fat; LBM, lean body mass.
* 3 g HMB free acid/d in three 1 g doses.
Baseline strength and body composition testing
At the start of the study, subjects reported to the laboratory for baseline one maximal repetition testing of the full squat, bench press and dead lift, and to determine their body composition on a GE Lunar Prodigy dual X-ray absorptiometry apparatus (software version, enCORE 2008). Each lift was deemed successful as described by the International Powerlifting Federation rules(Reference Gilbert and Lees19). Total body strength was calculated as the sum of the squat, bench press and dead lift one maximal repetition values.
Supplementation and resistance exercise protocol
Before the exercise session, subjects were randomly assigned to receive either 3 g/d of HMB-FA (combined with food-grade orange flavours and sweeteners) or a placebo (food-grade orange flavours and sweeteners) divided equally into three servings given 30 min before exercise and again with lunch and the evening meals. To ensure compliance, investigators watched as the subjects consumed the supplement before the exercise session. On the non-training days, subjects were instructed to consume one packet with three separate meals throughout the day. Empty packets were presented to the investigators upon returning to the laboratory following non-training days. All subjects participated in a high-volume resistance training session consisting of three sets of full squats, bench press, dead lifts, pull-ups, barbell bent over rows, parallel dips, military press, barbell curls and triceps extensions. Each exercise was performed for three sets of twelve maximal repetitions intensity, with a supervised and timed rest period length of 1 min between the sets. Training volume for the exercise session was calculated as the product of total sets, repetitions and weight lifted during each exercise.
Resting blood draws
Resting blood draws were obtained via venepuncture after a 12 h fast by a trained phlebotomist immediately before training and at 48 h after the resistance training bout, as this is when we have found that creatine kinase (CK) peaks in the blood(Reference Wilson, Kim and Lee16). Whole blood was collected and transferred into appropriate tubes for obtaining serum and plasma, and centrifuged at 1500 g for 15 min at 4°C. Resulting serum and plasma were divided into aliquots and stored at − 80°C until subsequent analyses.
Biochemical analysis
Samples were thawed once and analysed in duplicate for each analyte. All blood draws were scheduled at the same time of day to negate confounding influences of diurnal hormonal variations. Serum total testosterone, cortisol and C-reactive protein (CRP) were assayed via ELISA kits obtained from Diagnostic Systems Laboratories. All hormones were measured in the same assay on the same day to avoid compounded inter-assay variance. Intra-assay variance was less than 3 % for all analytes. Serum CK was measured using colorimetric procedures at 340 nm (Diagnostics Chemicals). Subjects were instructed to consume a meat-free diet 72 h before urine collection in order to prevent exogenous intake of 3-methylhistidine (3-MH). Urine collection occurred on two occasions. The first occurred the day before training. The second occurred 48 h following training. On both occasions, urine was collected for a period of 24 h. A 3-litre amber collection container and instructions were given to each subject before the urine collection period, and the subjects were instructed to start the collection after voiding their bladder in the morning and to continue for 24 h until voiding their urine the following morning. The subjects were instructed to keep the collection container refrigerated throughout the collection period. Urine volume was recorded, and a sample was taken and stored at − 80°C. Urinary 3-MH was determined from the 24 h urine collection by previously described methods (Metabolic Technologies)(Reference Rathmacher, Link and Flakoll20). Urinary creatinine was measured using a colorimetric Jaffe reaction (Cayman Chemical). The 3-MH data were expressed as a 3-MH:creatinine ratio.
Perceived recovery status scale
The perceived recovery status (PRS) scale was taken before and 48 h following the training session. The PRS score consists of values between 0 and 10, with 0–2 being very poorly recovered and with anticipated declines in performance, 4–6 being low to moderately recovered and expected similar performance and 8–10 representing high perceived recovery with expected increases in performance.
Diet control
Subjects could not have taken nutritional supplements for at least 3 months before data collection. At 2 weeks before and throughout the study, subjects were placed on a diet consisting of 25 % protein, 50 % carbohydrates and 25 % fat, which was designed by a registered dietitian who specialised in sport nutrition (Registered Dietitian, Licensed Dietitian/Nutritionist, and Certified Sports Nutritionist).
Statistical analysis
The treatment main effect was evaluated by an ANCOVA on all dependent variables. The mixed model in SAS (version 9.1; SAS, Inc.) was used to analyse the acute changes from baseline to 48 h. The baseline values were used as a covariate. For effect size, the Cohen statistic was calculated, and according to Cohen(Reference Cohen21), d values of 0·2, 0·5 and 0·8 represent small, medium and large effect sizes, respectively. An α of P< 0·05 was established a priori.
Results
Subject characteristics and total training volume
There were no differences between the groups for age, height, body weight, lean body mass or percentage of body fat (Table 1). The subjects studied were experienced in weight training and could be described as a muscular body type. There was no difference in total strength (sum of bench press, squat and dead lift one maximal repetition) between the groups, and total strength was 422 (sem 30) and 430 (sem 21) kg for placebo- and HMB-FA-supplemented groups, respectively. There was also no significant difference in total training volume (sets × repetitions × total weight lifted) completed for the exercise session between the groups (Table 1).
Changes in serum creatine kinase, perceived recovery status scale, urinary 3-methylhistidine, testosterone, free testosterone, cortisol and C-reactive protein
The high-volume exercise protocol resulted in a significant acute change in serum CK, which increased to a greater extent in the placebo (329 %) than in the HMB-FA group (104 %) (P= 0·004, d= 1·6; Fig. 1). Before the exercise session, serum CK levels were the same in placebo- and HMB-FA-supplemented subjects (141 (sem 14) and 158 (sem 16) IU/l, respectively). As a result of the exercise session, serum CK in the placebo group increased to 604 (sem 83) IU/l after 48 h while the HMB-FA-supplemented group showed a much diminished increase to only 322 (sem 35) IU/l (P= 0·004, d= 1·6). Supplementation with HMB-FA also resulted in an improved PRS score, an indication of a quicker recovery from the intense exercise session and potential for improved performance in subsequent sessions. The PRS score decreased from 9·1 (sem 0·4) to 4·6 (sem 0·5) 48 h after the exercise session in the placebo group, while in the HMB-FA group, the decrease was much less, decreasing from 9·1 (sem 0·3) to 6·3 (sem 0·3) from pre- to post-exercise (P= 0·005, d= − 0·48; Fig. 2). The PRS score indicated that the HMB-FA-supplemented subjects had recovered to a greater extent 48 h post-exercise than the placebo-supplemented group. While not significantly different between the groups, muscle protein breakdown, as measured by urinary 3-MH:creatinine ratio, remained constant in the placebo-supplemented group and approached a statistically lower rate of muscle protein degradation in the HMB-FA-supplemented group 48 h post-exercise (P= 0·08, d= 0·12; Table 2). There were no acute changes in plasma total or free testosterone, cortisol or CRP (Tables 2 and 3).
Fig. 1 Acute effects of β-hydroxy-β-methylbutyrate free acid (HMB-FA) on serum creatine kinase (CK). Resistance-trained males (n 20) were supplemented three times daily starting 30 min before an exercise bout with either HMB-FA (
, 1 g) or a placebo (
). HMB-FA significantly blunted the rise in CK (treatment effect P< 0·004). * Mean value was significantly different from baseline (P< 0·05). † Mean value was significantly different from the HMB-FA group (P< 0·05).
Fig. 2 Acute effects of β-hydroxy-β-methylbutyrate free acid (HMB-FA) on perceived recovery status (PRS) scale. Resistance-trained males (n 20) were supplemented three times daily starting 30 min before an exercise bout with either HMB-FA (, 1 g) or a placebo (). The PRS score consists of values between 0 and 10, with 0–2 being very poorly recovered and with anticipated declines in performance, 4–6 being low to moderately recovered and expected similar performance and 8–10 representing high perceived recovery with expected increases in performance. HMB-FA supplementation resulted in improved PRS after the exercise bout (treatment effect P< 0·005). * Mean value was significantly different from baseline (P< 0·05). † Mean value was significantly different from the HMB-FA group (P< 0·05).
Table 2 Effects of β-hydroxy-β-methylbutyrate (HMB) free acid on 3-methylhistadine and C-reactive protein (Mean values with their standard errors)
3-MH, 3-methylhistadine; CR, creatinine; CRP, C-reactive protein.
* Probability of treatment difference for the change from 0 to 48 h post-exercise.
Table 3 Effects of β-hydroxy-β-methylbutyrate free acid on anabolic and catabolic hormone profile (Mean values with their standard errors)
* Probability of treatment difference for the change from 0 to 48 h post-exercise.
Discussion
The primary aim of the present study was to determine the acute effects of HMB-FA administration in resistance-trained men on changes in serum CK, perceived recovery, anabolic and catabolic hormone status, protein breakdown and inflammation. The major findings of the present study were that HMB-FA sped recovery, blunted the decrease in PRS and may lead to decreased muscle protein breakdown.
Creatine kinase
CK is a commonly used indicator of myofibre disruption, which peaks 48 h post-resistance training(Reference Wilson, Kim and Lee16). As such, CK levels were assayed from blood draws before and 48 h after the resistance exercise protocol. There was a robust increase in serum levels of CK 48 h post-exercise, indicating the occurrence of muscle damage (Fig. 1). However, the present results demonstrated that an acute bolus ingestion of HMB-FA 30 min before exercise was able to attenuate the rise in CK. Previous studies that assessed muscle damage following a single exercise bout were conducted on untrained individuals who were administered HMB-Ca(Reference Wilson, Kim and Lee16, Reference van Someren, Edwards and Howatson22). Specifically, van Someren et al. (Reference van Someren, Edwards and Howatson22) found that 3 g HMB-Ca and 0·3 g α-ketoisocaproate administered daily for 2 weeks before an eccentric elbow flexor bout blunted the rise in CK, and that the supplementation also attenuated declines in one maximal repetition performance compared with placebo supplementation. Moreover, Wilson et al. (Reference Wilson, Kim and Lee16) demonstrated that a single 3 g serving of HMB-Ca administered 1 h before exercise in non-resistance-trained males prevented a significant increase in CK, lactate dehydrogenase and quadriceps soreness after an acute bout of exercise. The present study extends this research to a highly trained population, and utilised a more rigorous, multi-joint-centred resistance exercise bout.
Perceived recovery scale
Not permitting athletes adequate recovery time is detrimental to obtaining peak performance(Reference Kentta and Hassmen23, Reference Laursen and Jenkins24). Laurent et al. (Reference Laurent, Green and Bishop25) proposed a 0 (not recovered) to 10 (highly recovered) PRS scale based on the subjective physical and mental feelings of the athlete, as it pertains to their body before a training session. The PRS scale is an effective and valid tool for examining the recovery after a particular training session before a subject commences further training(Reference Laurent, Green and Bishop25). In the present study, subjects noted a significant drop in their PRS 48 h following the high-volume resistance training session (Fig. 2). However, this decline in perceived recovery was blunted by HMB-FA supplementation relative to placebo. Previous research with HMB had only analysed subjective measures of soreness(Reference Wilson, Kim and Lee16, Reference van Someren, Edwards and Howatson22). To our knowledge, we are the first to examine the supplement's effect on an athlete's mental perception of their physical readiness to train.
Muscle protein breakdown
One possible mechanism thought to underlie HMB's capacity to speed recovery is its effects on protein breakdown. Skeletal muscle mass is determined by two competing processes: protein synthesis and protein breakdown. In a catabolic situation, such as acute muscle injury, muscle protein breakdown is increased. As muscle protein is degraded, there is the release of the muscle-specific metabolite, 3-MH, which has been used as an index of myofibrillar protein breakdown(Reference Young and Munro26–Reference Elia, Carter and Bacon28). In the present study, the measure of urinary 3-MH:creatinine ratio approached significance, which may suggest a lower rate of muscle protein breakdown in the HMB-FA supplementation condition, compared with placebo supplementation. Previous research by Nissen et al. (Reference Nissen, Sharp and Ray7) investigated the effects of HMB during 3 weeks of resistance training. Their results found a decrease in protein breakdown at week 1, which was decreased even further by the end of week 2 with HMB-Ca supplementation. These findings may indicate a chronic effect of HMB supplementation on protein breakdown. It is important to note that there are non-skeletal muscle sources of 3-MH including gut tissues, which turn over at a more rapid rate than the significantly larger skeletal muscle pool. However, non-skeletal muscle sources do not significantly influence urinary 3-MH output even in extremely catabolic conditions. Furthermore, because the subjects consumed meat-free diets of the same composition and underwent the same workout protocol, it is unlikely that contributions from these other tissues significantly affected the present findings.
Testosterone, cortisol and C-reactive protein
Previous findings from Kraemer et al. (Reference Kraemer, Hatfield and Volek8) found that chronic supplementation with an HMB-containing supplement resulted in greater increases in resting levels of testosterone and decreases in cortisol levels. Conflicting results exist in untrained populations concerning the role of acute changes in hormones in neuromuscular adaptations(Reference West, Burd and Tang29, Reference Ronnestad, Nygaard and Raastad30). However, in trained populations similar to those in the present study, Hakkinen et al. (Reference Hakkinen, Pakarinen and Alen31, Reference Hakkinen, Pakarinen and Alen32) and Beaven et al. (Reference Beaven, Cook and Gill33) have demonstrated that resting and acute changes, respectively, in hormone status are correlated with changes in neuromuscular adaptations. In the present study, we examined testosterone (total and free), cortisol and CRP to determine any possible acute effects of HMB-FA on hormone status (anabolic and/or catabolic) and inflammation. The present results demonstrated no significant change in testosterone (free or total), cortisol or CRP levels from pre- to 48 h post-workout (Tables 2 and 3). Although some researchers have noticed changes in the levels of testosterone and cortisol in blood and saliva after resistance exercise(Reference Nunes, Crewther and Ugrinowitsch34–Reference Szivak, Hooper and Kupchak36), samples in those studies were taken either immediately or a few hours after exercise. Changes in hormone status may have occurred at time intervals different from the one chosen for the present study (48 h). Our post-exercise times for sampling were chosen because this is when maximal concentrations of muscle damage markers would be present in the blood. Previous research has indicated that CRP responds to chronic conditions of inflammation(Reference Milias, Nomikos and Fragopoulou37–Reference Goto, Takahashi and Yamamoto39), which may explain why the present results indicated no acute change in the inflammatory marker.
The present study, similar to others, has limitations. Primarily, we did not measure any indices of performance post-supplementation because the present study was designed to measure the acute effects of HMB-FA on muscle damage and recovery. Another limitation concerns our measure of CRP, which may be more indicative of long-term changes in inflammation. Changes in inflammatory markers with greater sensitivity to acute exercise may have yielded more insightful findings. Therefore, we suggest future research examine changes in performance post-supplementation. We also suggest future research sample classes of pro-inflammatory markers such as TNF-α, which is more sensitive to acute changes in inflammation(Reference Malm and Yu40).
Conclusions
The present study was the first to investigate the acute effects of an HMB-FA supplement in a resistance-trained population. The present results indicate that acute HMB-FA supplementation administered just before a bout of exercise attenuates serum indices of muscle damage and increases an athlete's mental perception of physical preparedness to train following high-volume, muscle-damaging resistance exercise. These findings suggest that athletes seeking to speed recovery from high-volume, high-intensity training can do so by consumption of HMB-FA 30 min before exercise.
Acknowledgements
This study was funded through a grant from Metabolic Technologies, Inc. HMB-FA was formulated and supplied by Metabolic Technologies, Inc. The authors' contributions are as follows: J. M. W., R. P. L. and J. M. J. were involved in the study design, training subjects, biochemical analysis of blood and manuscript preparation. J. R., S. M. B. and J. C. F. were involved in the study design, supplement preparation, analysis of 3-MH, data analysis and manuscript preparation. J. A. W., E. M. S. and N. M. D. were critical for data collection, training subjects and data input. J. R. S. and L. E. N. were critical for the study design and manuscript preparation. S. M. C. W. served as the study's sports dietitian and also assisted in the study design. J. M. W., R. P. L., J. M. J., J. A. W., N. M. D., E. M. S. and S. M. C. W. declare that they have no competing interests. J. C. F., S. M. B. and J. R. are employed by Metabolic Technologies, Inc.
