Subjects

Forty-five resistance-trained males who had been consistently training for at least one year volunteered for this study. Subjects were considered resistance trained if they had been consistently training for one year, a minimum of three days per week. Subjects were with 20.5 ± 3 yrs, 179 ± 7 cm, 84 ± 16 kg, and 17.3 ± 9% body fat. Subjects signed an informed consent that was approved by the Institutional Review Board for Human subjects prior to participation. Each subject completed a personal information sheet and a standard medical history form verified by a registered nurse. Subjects were free from any major metabolic disorders (i.e. heart disease, diabetes, thyroid disease, etc.) as well as major musculoskeletal disorders that would interfere with their ability to workout and/or complete the tests during the three testing sessions. Subjects were not permitted in the study if they had taken any self-reported ergogenic dietary supplements (i.e. creatine, androstendione, myostatin inhibitors, pro-hormones, etc.) within six months prior to the onset of the study. Subjects were asked to maintain their normal diet throughout the study and were not allowed to ingest any dietary supplement that contained potentially ergogenic nutrients. However, subjects were permitted to ingest energy-based sports drinks, energy bars, and protein powders provided that they did not contain any ergogenic nutrients. The reason for this was that many resistance-trained athletes ingest these energy-based supplements as a means to maintain their recommended dietary intake of calories. This intake was considered as part of their normal diet and was accounted for in analysis of dietary intake.

Experimental Design

The study was conducted as a double-blind, placebo controlled trial using parallel groups matched according to fat free mass. The independent variable was nutritional supplementation. Dependent variables included: dietary intake, body mass, body water (total, intracellular, extracellular) using bioelectrical impedance, body composition using dual energy x-ray absorptiometry (DEXA), upper and lower body 1 RM strength (bench press and leg press), muscle endurance (80% of 1 RM on bench press and leg press), anaerobic sprint power (Wingate cycle ergometer test), fasting clinical blood profiles (substrates, electrolytes, muscle and liver enzymes, red cells, white cells), and anabolic/catabolic hormones (free and active testosterone, cortisol).

Familiarization and Testing Sessions

Subjects were pre-qualified for entry into the study and then familiarized to the experimental design and practiced the exercise tests in order to get acquainted with the nature of the equipment and protocol prior to baseline testing. Subjects were scheduled for their first testing session and all questions and concerns were answered at this time.

Prior to baseline testing (T1), subjects recorded dietary intake on diet record forms for 4-days (three weekdays and one weekend day). Dietary records were analyzed by a registered dietitian using ESHA Research Inc. (Salem, OR) nutritional software. Subjects were instructed to abstain from exercise or physical activity for 48 hours prior to testing and were instructed to fast at least 10 hours prior to the baseline blood draw. On the day of baseline testing, subjects reported to the lab and completed questionnaires and information sheets. Height was measured using standard anthropometry and total body weight was measured using a calibrated electronic scale with a precision of ± 0.02 kg (Health-O-Meter, Bridgeview, IL). Whole-body composition was estimated by certified personnel using a Hologic QDR-4500W dual-energy x-ray absorptiometry (DEXA) using Hologic software version 9.80C (Waltham, MA). This test evaluates body composition and body density by scanning the entire body with a low dose of radiation. Test-retest reliability studies performed on male athletes with this DEXA machine yielded a mean deviation for total bone mineral content and total fat free/soft tissue mass of 0.31% with a mean intra-class correlation of 0.985. This method of determining body composition has been shown to be valid [15].

Following the DEXA, subjects donated approximately 20 ml of fasting blood from the antecubital vein in the arm via venipuncture using standard and sterile procedures. Blood was analyzed for basic clinical chemistry profiles for safety measures [glucose, protein, blood urea nitrogen (BUN), creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), gamma glutamyl transaminase (GGT), triglycerides, cholesterol] and whole blood cell counts [including hemoglobin, hematocrit, red blood cell counts, mean corpuscle volume (MCV), mean corpuscle hemoglobin (MCH), mean corpuscle hemoglobin concentration (MCHC), red cell dimension width (RDW), white blood cells (WBC), neutrophils, lymphocytes, monocytes, basophils, and eosinophils] by Quest Diagnostics (Dallas, TX). Blood serum samples were stored for later analysis of the anabolic/catabolic hormones (active testosterone, free testosterone, and cortisol) via assays in the Exercise and Biochemical Nutrition Laboratory.

Subjects first warmed-up (2 sets of 8 – 10 repetitions at approximately 50% of anticipated maximum) on the bench press. Subject's then performed successive 1-RM lifts starting at about 70% of anticipated 1-RM and increasing by 5 – 10 lbs until the subject reached their 1-RM. After the acquisition of max the participant rested five minutes and completed as many repetitions as possible at 80% 1-RM to assess muscular endurance. Subjects were instructed on proper technique and mechanics of the movement. Hand position was also recorded to ensure test re-test reliability. Subjects then rested for 10 minutes and warmed-up on the Nebula 45° Leg press (2 sets of 8 – 10 repetitions at approximately 50% of anticipated maximum). Subjects then performed successive 1 RM lifts on the leg press starting at about 70% of anticipated 1 RM and increasing by 10 – 25 lbs until reaching the subject's 1 RM. Subjects then perform an 80% of 1 RM endurance repetition tests on the hip/leg sled. Foot placement and sled height were recorded to ensure test re-test reliability. All strength testing was supervised by a Certified Strength and Conditioning Specialist (CSCS). Test re-test reliability of performing these strength tests on resistance-trained subjects in our laboratory have yielded low mean coefficients of variation and high reliability for the bench press (1.9%, intraclass r = 0.94) and leg press/hip sled (0.7%, intraclass r = 0.91).

Following the strength assessments and 10 minutes of rest, subjects then perform a 30-second Wingate anaerobic capacity test using a Lode computerized cycle ergometer (Groningen, Netherlands). Correlation coefficients of test-retest reliability for absolute peak power and mean power in our lab has been found to be r = 0.69 and r = 0.95, respectively. Subjects replicated all testing after 4 and 8 weeks of training and supplementation.

Supplementation Protocol

Subjects were matched into one of four groups according to fat free mass. Subjects were then randomly assigned to ingest in a double blind manner capsules containing a dextrose placebo (P); 800 mg/day of methoxyisoflavone (M) (MuscleTech Research & Development, Inc., Mississauga, ON); 100 mg/day of Polypodium Vulare/Suma root standardized for 30 mg of 20-hydroxyecdysone (E); or, 500 mg/day of sulfo-polysaccharides (CSP3) extracted from Cystoseira canariensis (Biotest Labs, Colorado Springs, CO). Subjects ingested the assigned capsules in the morning once per day for 8-weeks. The supplements were prepared in capsule form and packaged in generic bottles for double blind administration by MuscleTech Research & Development, Inc., (Mississauga, ON). Supplementation compliance was monitored by research assistants by having the subjects return empty bottles of the supplement at the end of 4 and 8 weeks of supplementation.

Training Protocol

Subjects participated in a periodized 4-day per week resistance-training program split into two upper and two lower extremity workouts per week for a total of 8-weeks. Prior to the workout, subjects performed a standardized series of stretching exercises as a warm-up. Subjects then performed an upper body resistance-training program consisting of nine exercises (bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flys, biceps curl, triceps press down, and abdominal curls) twice per week. Subjects also performed a seven-exercise lower extremity resistance-training program that could include (leg press, squat, back extension, step-ups, leg curls, leg extension, heel raises, and abdominal crunches) twice per week. The program was standardized at 3 sets of 10 repetitions with as much weight as they could lift per set (typically 60 – 80% of 1 RM) with no more than 2-minute rest periods between sets and no more than 3 minutes of rest between exercises. All training was conducted at the Student Life Center (SLC) at Baylor University. Subjects recorded the amount of weight lifted and number of repetitions performed for each set on training cards so that training volume could be determined. Subjects were also instructed to have their exercise card signed by SLC staff in order to verify attendance and completion of the workouts.

Blood and Serum Analysis

Two serum separation vacutainer tubes and one EDTA vacutainer tube was obtained from each subject. The serum vacutainers were separated via centrifugation a 6,000 rpm for 20 minutes. One serum separation tube and the EDTA tube were sent to Quest Diagnostics (Dallas, TX) for assay of a standard clinical chemistry profile and whole blood cell counts to ensure safety of supplementation during the protocol. The serum from the remaining separation tube was separated, labeled, and stored in micro centrifuge tubes at -80°C for later analysis. Following completion of the study, samples were analyzed for active testosterone, free testosterone, and cortisol with an enzyme immunoassay (EIA) assays using Goat-Anti-rabbit IgG (GARG) coated microplates. EIA kits were purchased from Diagnostic Systems Laboratories (Webster, TX). Assays were performed using a Jitterbug microplate shaker (Boekel Scientific-Philadelphia, PA) and a Tricontinent Multiwash Advantage microplate washer (Grass Valley, CA). The assays were run in duplicate and the absorbances of the standards, samples, and controls were determined at an optical density of 450 nanometers with a Wallac Victor[2] 1420 Multilabel counter by PerkinElmer (Boston, MA). Concentrations of active testosterone, free testosterone, and cortisol were expressed relative to changes in blood serum content. Intra-assay coefficients of variation were 5.3% and 6.8%, 7.5% and 5.4%, and 2.4% and 5.0%, respectively, for active testosterone (control I and II), free testosterone (control I and II), and cortisol (control I and II). Inter-assay coefficients of variation were 4.8% and 4.9%, 0.22% and 1.28%, and 12.0% and 6.1%, respectively, for active testosterone (control I and II), free testosterone (control I and II), and cortisol (control I and II).

Data Analysis

Data were analyzed using SPSS 11.5 for Windows (Chicago, Illinois). A repeated measures analysis of variances (ANOVA) was used for the analysis of the univariate means on the dependent variables and is presented in means and standard deviations. Delta scores (post and pre values) were calculated on selected variables and analyzed by ANOVA for repeated measures. Date were considered significant when the probability of Type I error was 0.05 or less. Significant group × time interactions were evaluated by least significant differences (LSD) post-hoc analysis to determine where the significance existed. Power analysis in a 4 × 3 design indicate that an n-size of 15 per group yields high power (>0.9) for delta values of 0.75 to 1.25. Data are presented as means ± SD changes from baseline.

Side Effects

Analysis of post study questionnaires revealed that subjects tolerated the supplementation protocol well with no reports of medical problems or symptoms.

Training Volume and Dietary Intake

Body Mass and Body Composition

Figure 1 presents mean changes in body mass and body composition for the P, M, E, and CSP3 groups. Training resulted in a statistically significant increase (p = 0.03) in DEXA fat free mass (lean/soft tissue mass plus bone mineral content) for all groups. However, no statistically significant differences were observed among the P, M, E, and CSP3 groups, respectively, in mean fat free mass (P 0.94 ± 1.5; M 0.29 ± 1.4; E -0.04 ± 1.3; CSP3 0.64 ± 1.4 kg, p = 0.40), fat mass (P0.69 ± 1.1; M 0.32 ± 1.6; E 0.69 ± 1.1; CSP3 -0.36 ± 0.7 kg, p = 0.17), percent body fat (P 0.59 ± 1.7; M 0.08 ± 1.7; E 0.70 ± 1.0; CSP3 -0.46 ± 0.8%, p = 0.18), or total body water (P 0.39 ± 3.3, M 0.16 ± 2.9, E 1.7 ± 2.5, CSP3 0.31 ± 2.4 L, p = 0.61).

Training Adaptations

Training resulted in statistically significant increase in 1-RM bench press (pre: 94.94 ± 22.8 to post: 100.84 ± 20.54 kg, p =< 0.0001) and 1-RM leg press (311 ± 76 to 341 ± 82 kg, p =< 0.0001) for all groups. No statistically significant interactions were observed among P, M, E, and CSP3 groups, respectively, in mean changes in bench press 1-RM (6.2 ± 7; 2.5 ± 5; 7.2 ± 10; 7.7 ± 6 kg, p = 0.29), leg press 1-RM (20.6 ± 33; 37.0 ± 39; 38.9 ± 45; 29.3 ± 36 kg, p = 0.63). Bench press lifting volume was significantly greater in the M group (105 ± 120; 102 ± 200; 124 ± 174; 25 ± 176 kg, p = 0.008) while leg press lifting volume was lower in the M group (97 6 ± 1,213; -799 ± 1,718; 1,259 ± 1,153; 978 ± 2,328 kg, p = 0.04). Likewise, no significant differences were observed in mean changes in Wingate peak power (82 ± 248; 30 ± 139; 28 ± 151; 17 ± 171 W, p = 0.82), Wingate mean power (20 ± 83; -13 ± 88; -18 ± 76; -15 ± 52 W, p = 0.55), or Wingate total work (582 ± 2,564; -216 ± 2,716; -420 ± 2,291; -528 ± 1,514 J, p = 0.29).

Blood Chemistry Profiles

No statistically significant differences were observed among groups as well as over time for the clinical chemistry profiles on variables including serum protein (p = 0.65), uric acid (p = 0.57), AST (p = 0.16), ALT (p = 0.56), GGT (p = 0.86), triglycerides (p = 0.62), and total cholesterol (p = 0.48). No statistically significant differences were observed among groups as well as over time in the whole blood cell count variables including hemoglobin (p = 0.74), hematocrit (p = 0.61), red blood cell counts (p = 0.39), MCV (p = 0.68), MCH (p = 0.20), MCHC (p = 0.29), WBC (p = 0.076), lymphocytes (p = 0.44), monocytes (p = 0.66), and eosinophils (p = 0.28) from whole blood analysis. However, significant changes were seen in neutrophils (2.05 ± 9.9; 4.4 ± 10.1; 4.3 ± 5.5, 5.8 ± 7.4 k/μL:, p = 0.04) and basophils (7.5 ± 7.6; 7.6 ± 25.1; 6.57 ± 16.1; 4.4 ± 7.8 k/μL, p = 0.04). All values remained within normal clinical ranges.

Anabolic/Catabolic Status

Table 2 presents selected markers of anabolic and catabolic status while Figure 4 &5 show changes in free and active testosterone for the P, M, E, and CSP3 groups. No statistically significant differences were observed among P, M, E, and CSP3 groups, respectively, in changes in total testosterone (0.21 ± 0.83, 0.26 ± 1, 0.02 ± 1.21, 0.10 ± 0.93 ng/ml, p = 0.67), free testosterone (4.39 ± 10.4, 5.54 ± 6.85, 1.39 ± 11.84, 1.77 ± 8.62 pg/ml, p = 0.25), cortisol (2.68 ± 2.46, 1.11 ± 6.3, 1.93 ± 5.57, 1.66 ± 10.54 μg/dl, p = 0.57), total testosterone to cortisol ratio (0.05 ± 0.09, 0.02 ± 0.16, 0.06 ± 0.17, 0.02 ± 0.16, p = 0.43). In addition, no differences were observed from pre to post testing for all variables in Table 2.