Figure 1 Schematic representation of experimental protocol. Participants followed their normal diet and completed 7-day food diaries during the familiarization and pre supplementation weeks and were asked to replicate their training regimes this website throughout the study period. The diet was analyzed for energy intake and macronutrient content using the CompEat nutritional analysis software, which is based on the UK, integrated database, McCance and Widdowson’s [15]. Participants were asked to avoid

caffeine intake and alcohol for the full length of their participation in the trial to lessen any possible confounding effects of caffeine on Cr [13]. Experimental procedures: total body water determination Participants were required to report to the laboratory

before breakfast after an 8 h fast. Measurements of TBW using both BIA (Bodystat Multiscan 500, Bodystat Ltd, Isle of Man, UK) and D2O method were carried out. Briefly, BIA is an non-invasive method that involves placing two current-inducing electrodes and two detector electrodes on the dorsal surfaces of the right hand and MK-0457 in vivo foot and a small (and imperceptible) electrical current (500 Micro-Amps) introduced between these. On arrival to the laboratory, participants provided a baseline urine sample and were then asked to lie comfortably DCLK1 in a supine position while a 21 G cannula was introduced into a superficial vein on the dorsal surface of the participant’s arm. Blood samples (10 mL) were taken before and after the re-breathing procedure [16–18]. Participants were then asked to orally ingest D2O (Ontario hydro, Canada). The validity of method has been previously assessed [19]. Each participant was given an oral dose of 0.5 g.kg-1 BM of D2O in the morning after a baseline urine sample has been collected. To evaluate the volume of isotopic distribution in body water, a urine sample was collected again after 6 h, in a dry plastic container. Participants

were instructed to empty their bladder completely at 5 h post D2O ingestion and were allowed breakfast, a light lunch as well as to pass urine and drink as normal within the 6 h period. For purposes of analysis, the investigator transferred 2 mL from all urine samples from the dry plastic containers to glass vessels and stored in −20°C. Urine samples were then analyzed by an LY2874455 isoprime isotope ratio mass spectrometer (Elementar Ltd, Manchester, UK), coupled to a Eurovector gas chromatograph (GC) fitted with an HT300A autosampler, as described elsewhere [20]. Experimental procedures: Analyses of total haemoglobin mass Briefly, a bolus of chemically pure CO dose of 1.0 mL.kg-1 BM was administered with the first breath through a spirometer and rebreathed for 2 min with 4 L of oxygen.