Subjects
Twelve healthy, recreationally active men (mean ± SD age, 21 ± 3 yr; mass, 79.1 ± 10.7 kg) participated. A within-treatment experimental design was used to increase sensitivity and reliability of measures and thus, each subject acted as his own control. Subjects were matched according to age, body size, and training experience prior to their initial random placements into one of the two treatment conditions. Eligibility required at least three months of resistance training experience including the squat exercise. Medical histories were obtained to exclude medical, musculoskeletal, and endocrine disorders, concurrent nutritional supplementation, and anabolic drugs. All subjects were informed of the benefits and potential risks of the investigation and signed a University Institutional Review Board approved consent form for recruitment and participation.
Study design
A balanced, randomized, double blind, repeated-measures, placebo, cross-over design was used. All subjects performed a testing protocol providing direct data on physical performance. Recovery effects were measured by repeating this testing protocol 24 hr following this first visit. After this initial (baseline) testing, subjects underwent 14 days of betaine or placebo supplementation again followed by exercise testing on two consecutive days. Subjects underwent a 14 day washout period and then crossed over into the other 14-day period of either betaine or placebo supplementation. In addition to performance testing, some blood variables were measured, and special attention was given to dietary and activity control among and within subjects. Subjects refrained from any exercise for 48 hr prior to the scheduled performance testing sessions. All testing sessions were conducted between 0700 and 1000 hr, but at the same time of day for each respective subject. A standardized whole-body resistance training session was performed twice (mid-week) during the 14-day supplementation periods to maintain the subjects' level of conditioning.
Betaine supplementation
Betaine supplement (B) was given as 1.25 grams (g) of betaine (Danisco Inc., Ardsley, NY) in 300 mL of Gatorade© sports drink, taken twice daily at standardized times for each subject. Additionally, on each testing day subjects received a morning dose of the betaine supplement or placebo. Placebo (P) drinks were the same sports drink formulation and flavor without the betaine additive. Researchers involved in data collection and participants themselves were blinded to treatment until an un-blinded outside researcher revealed treatments following study completion.
Exercise testing protocol
After a standardized warm up of 5 minutes of low intensity cycling, subjects performed the following high intensity strength/power resistance exercise challenge (REC).
4 sets × 3 repetitions Vertical Jump
2-minute rest following each set
Maximal effort Isometric Squat (lasting 6-10 sec)
2-minute rest
3 repetitions Squat Jump @ 30% 1 RM
2-minute rest
3 sets Back Squat @ 85% 1 RM until fatigue
2-minute rest following each set
Maximal effort Isometric Bench Press (lasting 6-10 sec)
2-minute rest
repetitions Bench Throw @ 30% 1 RM
2-minute rest
3 sets Bench Press @ 85% 1 RM until fatigue
2-minute rest following each set
Standardized resistance exercise testing protocols are commonly used in our laboratory for research studies [e.g. [10, 11]]. During this protocol, measures of power (W) and force (N) were measured using a force plate (AccuPower, Athletic Republic, Fargo, ND, USA).
Blood variables
Blood samples were collected via an indwelling catheter placed in the antecubital forearm vein at the beginning of each day of exercise testing. Samples were obtained before exercise testing began, immediately following vertical jump, following squat testing, immediately post all exercise testing, and fifteen minutes following cessation of exercise, for a total of five blood timepoints. After whole blood analyses, blood plasma was obtained via centrifugation (Hettich Centrifuge, Beverly, MA) at 3200 RPM, 4°C, 20 minutes, and stored at -80°C until further analysis. Betaine was analyzed in EDTA preserved plasma samples. High performance liquid chromatography was utilized with a silica column in a mixed partition and ion exchange mode following a method previously described [12]. Hematocrit (International Equipment Co., Needham Heights, MA, microcapillary reader) and hemoglobin concentration (Hemocue 201+ Analyzer, Lake Forest, CA) were obtained from whole blood, plasma osmolality was measured with an osmometer (Advanced Instruments, Inc., Norwood, MA, Model 3250) prior to sample storage. Glucose and lactate concentrations were analyzed using a glucose/lactate analyzer (2300 YSI Stat Plus, Yellow Springs, OH). All blood variables were measured in respective SI units.
Other variables
Subjects submitted self-administered 3-day diet records and six week activity records to verify consistency in diet and activity during study participation. Urine specific gravity (USG) (ATAGO clinical refractometer, Cole-Parmer, Vernon Hills, IL), osmolality, and body mass were measured prior to each exercise testing session to verify hydration status.
Statistical analysis
All variables were analyzed using Repeated Measures ANOVA with supplement treatment (placebo or betaine, two levels) and the appropriate number of time points as within subject factors. The sphericity assumption was met and significance was set at p < 0.05. Post hoc comparisons were t tests with Bonferroni corrections applied. The main effects of supplement were evaluated in the statistical model, and time effect and supplement × time interaction effects were also evaluated. Data are presented as means ± standard deviation for all variables.