Permeabilized skeletal muscle fibers were immediately prepared from the muscle tissue collected in the preservation medium, as described elsewhere (4 (link),23 (link)). Subsequently, the permeabilized muscle fibers (∼2.5 mg wet weight) were analyzed for mitochondrial function using an oxygraph (OROBOROS Instruments, Innsbruck, Austria), in essence according to Phielix et al. (4 (link)). To prevent oxygen limitation, the respiration chambers were hyperoxygenated up to ∼500 μmol/l O2. Subsequently, two different multisubstrate/inhibition protocols were used in which substrates and inhibitors were added consecutively in saturating concentrations. State 2 respiration was measured after the addition of malate (4 mmol/l) plus octanoyl-carnitine (50 μmol/l) or malate (4 mmol/l) plus glutamate (10 mmol/l). Subsequently, an excess of 2 mmol/l of ADP was added to determine coupled (state 3) respiration. Coupled respiration was then maximized with convergent electron input through Complex I and Complex II by adding saturating concentrations of succinate (10 mmol/l). Finally, the chemical uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) was titrated or oligomycin (2 μg/ml) was added to evaluate the maximal capacity of the electron transport chain and the respiration not coupled to ATP synthesis (state 4o respiration), respectively. The integrity of the outer mitochondrial membrane was assessed by the addition of cytochrome C (10 μmol/l) upon maximal coupled respiration. All measurements were performed in duplicate.