Twenty-four male subjects participating in regular high-intensity resistance training (4–6 times per week) were recruited for this study and provided their written, informed consent. The study conformed to the standards set by the Declaration of Helsinki, and carried the approval of the Human Research Ethics Committee of RMIT University and the Australian Institute of Sport.
After reporting to the laboratory to undergo a whole-body DEXA scan and to determine one-repetition maximum (1-RM) bilateral knee extensor strength, subjects were provided with a standardized diet for the 72h prior to the trial that provided an energy availability of 45 kcal/kg fat-free mass with a macronutrient contribution 1.5 g protein/kg/d and 4 g carbohydrate/kg/d , respectively. Subjects were instructed to refrain from training and other vigorous physical activity during the 72h period.
Subjects reported to the laboratory after a 10-h overnight fast and provided a spot urine sample to determine baseline enrichment (see below). Following two warm up sets (5 repetitions at 60% and 70% 1-RM), subjects completed an acute bilateral leg extension exercise session (4x10 sets at 80% 1-RM with 3 min recovery between sets). Subjects were randomly allocated to receive a total of 80g of whey protein isolate over 12h in one of three different ingestion patterns: pulsed feeding (PULSE), 8x10g every 1.5h; intermediate feeding (INT), 4x20g every 3h ; or bolus feeding (BOLUS), 2x40g every 6h. The BOLUS group was designed to simulate the ingestion of 2 large meals of the three meals one might consume in a given day. The first beverage of each group was consumed immediately after exercise and included 200mg of [15N]glycine. Subjects rested comfortably in the laboratory and collected all urine over the complete 12-h period, which was pooled and stored at −80°C until further analysis.
Urinary ammonia was isolated by cation exchange resin with the 15N]enrichment determined by isotope ratio mass spectrometry. Whole-body protein turnover (Q) was calculated using the 15N]ammonia end-product method as described previously, which provides consistent rates over 12h. Concentrations of urinary urea and creatinine, the major nitrogen containing metabolites in urine, were measured by automated analyser at the Laboratoire Central de Chimie Clinique (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland). Whole-body protein synthesis (S) and protein breakdown (B) were calculated as previously described with estimated fecal and miscellaneous nitrogen losses of 0.95g/12h and 0.69g/12h, respectively, which were based on half the daily excretion previously measured in strength athletes consuming a moderate protein diet. Whole-body net protein balance (NB) was calculated as: S minus B. Data were expressed normalized to both whole body mass (BM) and fat- and bone-free (i.e., lean) body mass (LBM).
Data were analyzed using a one-way repeated measure analysis of variance (ANOVA) with Student Newman Keuls post-hoc analysis (Sigmastat V3.11). In the event of non-normal distribution, data were log-transformed prior to analysis. Statistical significance was established at P<0.05 and all data are expressed as mean ± standard deviation. It has been demonstrated previously that the 15N]glycine-measured increase in NB is able to reasonably predict the training-induced increase in lean body mass in young men. To account for possible subtle differences in the most physiologically meaningful variable of NB, mean effect sizes and 90% confidence intervals (CI) were also calculated to allow for probabilistic magnitude-based inferences between groups[18, 19]. Quantitative chances of benefit and harm were assessed according to previously published cut-points using the smallest standardized change in the mean, as described in detail elsewhere[18, 19].