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Glass capillary tubes were used to collect 50 ��l of blood during the GXT (D957G-70-35; Clinitubes, Radiometer, Copenhagen, Denmark) and 100 ��l of blood during the HIE and training sessions (D957G-70-125; Clinitubes, Radiometer). A hyperaemic ointment (Finalgon; Boehringer Ingelheim, Ingelheim, Germany) was applied to the earlobe 5�C7 min prior to initial blood sampling, and all blood was sampled from the earlobe. Capillary blood samples were taken at rest and immediately following each 4 min stage of the GXT, and at rest and immediately after the HIE. Plasma pH and [BLa] were determined using a blood-gas analyser (ABL 625; Radiometer). The blood-gas analyser was regularly calibrated using precision [http://www.selleckchem.com/products/i-bet-762.html I-BET-762 in vivo] standards and routinely assessed by external quality controls. On the day of the HIE-only cycle test, two incisions were made under local anaesthesia (5 ml, 1% Xylocaine) into the vastus lateralis of each subject. The first incision was used [http://en.wikipedia.org/wiki/Diglyceride diglyceride] for the pretest biopsy and then subsequently used for the post-test biopsy; pre- and post-test biopsy samples were taken with the needle inserted at different angles. The third biopsy sample was taken from the second site. All biopsies were performed with manual suction applied, and postbiopsy the whole needle was rapidly submerged in liquid nitrogen. The first muscle sample was taken prior to warm up, during supine rest. The second muscle sample was taken immediately following the cessation of the HIE ([http://www.selleckchem.com/products/iox1.html IOX1 supplier] PCr resynthesis postexercise is rapid (Edge et al. 2006a) and because few studies have examined changes in the recovery of muscle metabolites over such a time frame. The subject remained on the cycle ergometer for all postexercise testing. Pre- and post-training samples were taken from opposite legs, at approximately the same position. The samples were then removed from the biopsy needle, rapidly frozen in liquid N2 and stored at ?80��C until subsequent analysis. Muscle Na+,K+-ATPase content was determined in quadruplicate using the vanadate-facilitated [3H]oubain binding site content (Norgaard et al. 1984). Muscle samples were cut into 2�C5 mg pieces and washed twice for 10 min in 37��C vanadate buffer containing 250 mm sucrose, 10 mm Tris, 3 mm MgSO4 and 1 mm NaVO4 (pH 7.2�C7.4). Muscle samples were then incubated for 120 min at 37��C in the vanadate buffer with the addition of 3H (10�C6 m, 2.0 mCi ml?1).