Extracts were assayed for Cr in the presence

of 50 mM imidazole buffer, pH 4.7; 5 mM magnesium chloride; 20 mM potassium chloride; 25 μM phosphoenolpyruvate; 200 μM ATP; 45 μM NADH; 1250 U/mL lactate dehydrogenase; 2000 U/mL pyruvate kinase. The assay was carried out in a standard fluorescence microplate reader using 10 μL of sample to 1 mL of reagent. The reactant solution was vortexed and read using a fluorometer (Shimadzu RFMini 150, Japan) with R406 nmr an excitation wavelength of 340 nm and an emission wavelength of 460 nm for baseline absorbance values. Five μL of CK (25 μ/mg) was added to 1 mL of the above buffer and stabilized using 1 mL of reagent. After 10 minutes the plate was read again for post-reaction absorbance values. Test to test reliability of duplicate muscle creatine assays was 0.22 ± 2.4% (r = 0.90) with a coefficient of variation of 6.8. We also assayed muscle samples for phosphocreatine (PCr) but several values were out of normal ranges, there was large variability in values observed,

and overall PCr levels declined over time despite creatine supplementation suggesting a lack of validity in this assay. Therefore, these data were not reported. Performance tests Maximal strength tests were performed using a standard isotonic Olympic bench press and hip sled/leg press (selleck chemicals llc Nebula Fitness, Versailles, OH) according to standardized procedures [44]. Hand positioning on the bench press and foot and seat position on the hip sled/leg press were standardized between trials. Participants followed a standardized warm-up https://www.selleckchem.com/products/kpt-330.html (10 repetitions at 50% of 1RM) Bacterial neuraminidase prior to beginning 1RM attempts. Rest recovery was standardized between attempts at 2-min and participants typically reached their 1RM within 3–5 attempts after warming up. Participants performed the hip sled/leg press 1RM test, rested for 4 minutes, and then began warming up on the bench press. Bench press 1RM was

determined following similar procedures as the hip sled/leg press 1RM test. Test-to-test reliability of performing these tests in our lab on resistance-trained participants have yielded low day to day mean coefficients of variation and high reliability for the bench press (1.1%, intra-class r = 0.99) and hip sled/leg press (0.7%, intra-class r = 0.91). Subjects rested for about 20-minutes and then warmed up on a bicycle ergometer for 3-minutes (70 rpm @ 1 kg resistance). Participants then performed a 30-second Wingate sprint anaerobic capacity test on a Lode Excalibur Sport 925900 cycle ergometer (Lode BV, Groningen, The Netherlands) at a standardized work rate of 7.5 J/kg/rev. The seat position was standardized between trials and the participant was asked to pedal as fast as possible prior to application of the workload and sprint at all-out maximal capacity during the 30-second test.