The L-alanyl-L-glutamine supplement (0.2 g·kg-1 or 0.05 g·kg-1 body mass per liter) marketed as “”Sustamine™”" (Kyowa Hakko USA, Cell Cycle inhibitor New York, NY) was mixed with water and was indistinguishable in appearance and taste from the placebo. Time to Exhaustion Test After the dehydration and rehydration phase, subjects began the exercise protocol. Subjects exercised at a workload that elicited 75% of their on a cycle ergometer. Subjects were encouraged to give their best effort during each

trial, and were verbally encouraged throughout each exercise trial. , RER, , RER, and HR, were measured continuously. HR and blood pressure (BP) were recorded before and at the conclusion of exercise. Time to exhaustion was determined as the time that the subject could no longer maintain the workload and/or reached volitional exhaustion. Blood Measures A baseline (BL) blood draw occurred during T1. No other blood was drawn during that trial. The BL blood sample was drawn following a 15-min equilibration period prior to exercise. All day of trial blood samples (DHY, RHY and IP) were

obtained using a 20-gauge Teflon cannula placed in a superficial forearm vein using a 3-way stopcock with a male luer lock adapter. The cannula was maintained patent using an isotonic saline solution (with 10% heparin). During trials T2 – T5 blood draws occurred once goal body mass was achieved (DHY), immediately prior to the exercise stress (RHY) and immediately following the exercise protocol (IP). IP blood samples were taken within 15 seconds of exercise cessation. Subjects returned to the selleck compound laboratory JNJ-64619178 cell line 24-h post-exercise for an additional blood draw (24P). All BL and 24P blood samples were drawn with a plastic syringe while the subject was in a seated position. These blood samples were obtained from an

antecubital arm vein using a 20-gauge disposable needle equipped with a Vacutainer® tube holder (Becton Dickinson, Franklin Lakes, NJ) with the subject in a seated position. Each subjects’ blood samples were obtained at the same time of day during each session. Blood samples were drawn into plain or EDTA treated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Blood Bumetanide samples were analyzed in triplicate for hematocrit via microcapillary technique and hemoglobin via the cyanmethemoglobin method (Sigma Diagnostics, St. Louis, MO). The remaining whole blood was centrifuged for 15 min at 1500 g at 4°C. Resulting plasma and serum were aliquoted and stored at -80°C until analysis. Samples were thawed only once. Biochemical and Hormonal Analyses Serum testosterone (TEST), cortisol (CORT) and growth hormone (GH) concentrations were determined using enzyme immunoassays (EIA) and enzyme-linked immunosorbent assays (ELISA) (Diagnostic Systems Laboratory, Webster, TX). Serum aldosterone (ALD) and IL-6 concentrations were determined using an EIA assay (ALPCO Diagnostics, Salem, NH).