The current study was a randomized, double-blind, placebo- and diet-controlled experiment consisting of a 12-week-periodized resistance-training program. The training protocol was divided into three phases and consisted of a non-linear-periodized resistance-training program for the first 8 weeks, followed by a 2-week overreaching cycle, and finally a 2-week taper of the training volume (Tables S1–S3). Muscle mass, body composition, strength, power, resting plasma testosterone, cortisol, and creatine kinase (CK) levels were examined collectively at the end of weeks 0, 4, 8, and 12 to assess the chronic effects of HMB; these measures were also assessed at the end of weeks 9 and 10, corresponding to the mid- and endpoints of the phase 2 overreaching cycle. The study was approved by the University of Tampa Institutional Review Board and registered with ClinicalTrials.gov (NCT01508338).
Twenty-four resistance-trained males were tested and included in the study. Subjects were organized into quartile blocks based on their LBM and strength. Following this, each quartile was randomized to one of the treatment groups using computer-generated random numbers. After treatment assignment, the groups were assessed to confirm there were no differences between groups. Of those randomized to the treatments, three subjects dropped from the placebo group, two because of injury and one because of the time commitment, while one dropped from the HMB-FA group due to injury. All drop outs occurred during the first 4 weeks of the study. The remaining 20 subjects (21.6 ± 0.5 years of age) consisting of nine placebo (87.1 ± 4.8 kg; 180.9 cm) and 11 supplemented (83.1 ± 2.8 kg; 179.0 ± 2.1) males with an average squat, bench press, and deadlift of 1.7 ± 0.04, 1.3 ± 0.04, and 2.0 ± 0.05 times their bodyweight, respectively, completed the study. There were no significant differences for age, body weight, height, or BMI between the treatment groups at the start of the study. Participants were excluded if they were currently taking anti-inflammatory agents as well as any other performance-enhancing supplement, or if they smoked. The participants must not have taken any nutritional supplements for at least 3 months prior to the start of data collection. Each participant signed an informed consent approved by the University of Tampa Institutional Review Board before participating in the study in the Department of Health Sciences and Human Performance at the University of Tampa, Tampa, FL, USA.
Strength, power, body composition, and skeletal muscle hypertrophy testing
Strength was assessed via a one-repetition maximum (1-RM) testing of the back squat, bench press, and deadlift. The intraclass correlation coefficient (ICC) for the test–retest of strength in the squat, bench press, and deadlift ranged from r = 0.956 to 0.982. Body composition [LBM, fat mass (FM), and total mass] was determined on a Lunar Prodigy dual X-ray absorptiometry (DXA) apparatus (software version, enCORE 2008, Madison, WI, USA). Tests for the DXA were performed at the same time of day in a fasted state and the ICC was r = 0.981. Skeletal muscle hypertrophy was assessed via changes in ultrasonography (GE Logiq e 2008, Wauwatosa, WI, USA) determined combined muscle thickness of the vastus lateralis (VL) and vastus intermedius (VI) muscles. The ICC for the test–retest of muscle thickness measurements was r = 0.975.
Peak power (PP) was assessed during maximal cycling (modified Wingate test) and jumping movements. During the cycling test, the volunteer was instructed to cycle against a predetermined resistance (7.5 % of body weight) as fast as possible for 10 s (Smith et al. 2001) on a cycle ergometer (Monark model 894e, Vansbro, Sweden). Wingate PP was recorded using Monark anaerobic test software (Version 1.0, Monark, Vansbro, Sweden). From completion of Wingate tests performed over several days, the ICC for Wingate PP was 0.966.
Measurements of PP for the vertical jump were also taken on a multicomponent AMTI force platform (Advanced Mechanical Technology, Watertown, MA USA) which interfaced with a personal computer at a sampling rate of 1,000 Hz (Lowery et al. 2012). Data acquisition software (LabVIEW, version 7.1; National Instruments Corporation, Austin, TX, USA) was used to calculate vertical jump PP. The ICC for VJ PP was 0.971.
Supplementation, diet control, and exercise protocol
Prior to the study, participants were randomly assigned to receive either 3 g per day of HMB-FA or a placebo divided equally into three servings. Each serving was formulated with 1 g of HMB-FA. The first serving was given 30 min prior to exercise and the remaining two servings given with the mid-day and evening meals. On the non-training days, participants were instructed to consume one serving with each of three separate meals throughout the day. Blinding occurred via an outside researcher who sent an isocaloric supplement and placebo in identical looking and flavored packets containing either 3 g per day of HMB-FA (combined with food-grade orange flavors and sweeteners) or a placebo (corn syrup combined with food-grade orange flavors and sweeteners) divided equally into three servings daily. This researcher was not involved in direct data collection, or statistical analysis, and did not meet any of the subjects. For this reason, neither the researchers conducting the study, nor the subjects knew which participant was assigned to which group. Moreover, the code was not broken until after all of the data were entered into a computer spreadsheet, and sent to an outside researcher who was also blinded to the treatment groups. The supplementation was continued daily throughout the training and testing protocols. Two weeks prior to and throughout the study, participants were placed on a diet consisting of 25 % protein, 50 % carbohydrates, and 25 % fat by a registered dietician (M.S., RD, LD) who specialized in sports nutrition. The participants met as a group with the dietitian, and they were given individual meal plans at the beginning of the study. Diet counseling was continued throughout the duration of the study. Assessment of 3-day food records taken at the beginning, mid, and last week of the study revealed that diets consisted of 22 % protein, 45 % carbohydrates, and 33 % fat, with no differences between groups. Compliance of supplementation was assessed by having the participants hand their empty packets to a researcher at the beginning of each training day. Compliance was over 98 % for supplementation.
The purity of HMB-FA was determined by the manufacturer (TSI, Missoula, MT, USA) using high-pressure liquid chromatography to be 99.7 %. The primary impurities were acetate and water. Metabolic Technologies Inc. (MTI, Ames, IA, USA) independently assayed the HMB-FA for purity and confirmed these results. In addition, MTI assayed HMB-FA for dehydroepiandrosterone (DHEA) a contaminant which has been found in nutritional supplements using gas chromatography–mass spectrometry (Thuyne and Delbeke 2005). DHEA was not detected in the HMB-FA (<1 ng/g). In addition, a sample was sent to an independent laboratory (MVTL, New Ulm, MN, USA) for microbial and heavy metals testing. HMB-FA tested negative for E. coli, Listeria, and Salmonella. Copper, zinc, calcium, mercury, cadmium, and lead were less than the instrument’s detection limits and arsenic was detected at 37 PPB.
The training was divided into three phases, with Phase 1 (Table S1) consisting of a daily undulating periodized resistance-training program 3 days per week during weeks 1 through 8. This protocol was modified from Kraemer et al. (2009). Phase 2 (Table S2) consisted of a 2-week overreaching cycle during weeks 9 and 10 wherein participants resistance trained 5 days per week and also performed an additional day of Wingate and power testing. Finally, phase 3 (Table S3) consisted of a tapered training volume for weeks 11 and 12. All training sessions were monitored and controlled by the researchers, and if a session was missed at a specific time of day it was made up within 24 h. Using this criterion, compliance was 100 % for all subjects who completed the study.
Resting blood draws
All blood draws throughout the study were obtained via venipuncture after a 12 h fast by a trained phlebotomist. All blood draws were scheduled at the same time of day to negate confounding influences of diurnal hormonal variations. Whole blood was collected and transferred into appropriate tubes for obtaining serum and plasma and centrifuged at 1,500 g for 15 min at 4 °C. Resulting serum and plasma were then aliquoted and stored at −80 °C until subsequent analyses. A portion of the blood samples taken at weeks 0, 4, 8, and 12 were used for measurements (Any Lab Test Now®, Tampa, Fl, USA) of glucose, blood urea nitrogen, creatinine, eGFR, Na+, K+, Cl−, CO2, Ca2+, protein, albumin, globulin, albumin:globulin ratio, total bilirubin, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase. A complete blood count was also performed on each blood sample. In addition, a urinalysis (urine specific gravity, pH, and Urobilinogen) was conducted on a sample of urine. The samples were submitted to an outside laboratory for analysis (Any Lab Test Now®, Tampa, FL, USA) at weeks 0, 4, 8, and 12.
Samples were thawed one time and analyzed in duplicate for each analyte. Serum total and free testosterone, cortisol, and C-reactive protein (CRP) were assayed via ELISA kits (Diagnostic Systems Laboratories, Webster, TX, USA). All hormones were measured in the same assay on the same day to avoid interassay variation. All samples were analyzed in duplicate. The intra-assay variance was calculated by coefficient of variation [(SD/mean) × 100]. Intra-assay variance was determined to be <3 % for all analytes. Serum CK was measured at 340 nm using colorimetric procedures (Diagnostics Chemicals, Oxford, CT, USA). As a measure of muscle protein degradation, the ratio of urinary 3-methylhistidine to creatinine (3MH:Cr) was calculated. The participants were instructed to consume a meat-free diet for 72 h prior to each measurement period at weeks 8, 9, and 10. Urine was collected for 24 h, uniformly mixed, sampled, and then stored at −80 °C until subsequent analyses. The 3MH was measured using a previously described GC/MS method (Rathmacher et al. 1992). Urinary creatinine (Cr) was measured using a colorimetric Jaffe’s reaction (Cayman Chemical, Ann Arbor, MI, USA). The 3MH:Cr ratio over the 24-h period was then calculated.
Perceived recovery status scale
Perceived recovery status (PRS) scale was measured at weeks 0, 4, 8, 9, 10, and 12 to assess subjective recovery during the training phases. The PRS scale consists of values between 0 and 10, with 0–2 being very poorly recovered with anticipated declines in performance, 4–6 being low to moderately recovered with expected similar performance, and 8–10 representing high perceived recovery with expected increases in performance. The PRS scale has been demonstrated as a valid cognitive indicator of performance and fatigue (Laurent et al. 2011; Sikorski et al. 2013).
A one-way ANOVA model was used to analyze the baseline characteristic data using the Proc GLM procedure in SAS (SAS Institute, Cary, NC, USA). The primary outcome measure of this double-blind, placebo- and diet-controlled randomized intervention study was muscular strength and power. The secondary outcome measure was muscle hypertrophy. The main effect of treatment (Trt) was included in the model. Changes over the 12-week study were analyzed by repeated measures ANOVA using the Proc Mixed procedure in SAS with the initial week of the period, week 0, used as a covariate and the main effects were Time, Trt, and Trt × Time. In addition, the overreaching phase of the study was further assessed using a repeated measures ANOVA with Proc Mixed procedure in SAS with the week 8 time point used as a covariate and the main effects were Time, Trt, and Trt × Time. Least squares means procedure was then used to compare treatment means at each time point. The n size was based on a power analysis of LBM differences found by Kraemer et al. (2009). Statistical significance was determined at p ≤ 0.05. The results are presented as mean ± standard deviations.