Cytochrome-c oxidase activity assay.

To confirm that the physical activity protocol elicited a training effect, we measured cytochrome-c oxidase (COX) activity in the gastrocnemius muscles. COX activity was determined according to a modification of a method previously described (8). Briefly, cross-sections of mixed gastrocnemius muscles (from the midsection of the muscle belly), weighing roughly 20–30 mg, were diluted 80-fold (sedentary) or 160-fold (physical activity) in extraction buffer (100 mM Na-K-Phosphate, 2 mM EDTA, pH 7.2). Muscle extracts were prepared by homogenization with metal beads (2 × 30 s) at a frequency of 30 Hz in a magnetic homogenizer (Mixer Mill MM 400, Retsch, Germany). These homogenates were then used for the analysis of the maximum rate of oxidation of fully reduced cytochrome c at 30°C as indicated by changes in absorbance (550 nm).