The aim of the present work was to test the hypothesis that moderate endurance treadmill training ameliorates gastrocnemius mitochondrial bioenergetics and increases the tolerance to the calcium-induced mitochondrial permeability transition pore (MPTP) opening. Twelve adult (6-8 week old) male Wistar rats were randomly divided into two groups (n=6per group): sedentary and trained (14 week of endurance treadmill running, 60min/day). Several end-points for invitro gastrocnemius mitochondrial function including oxygen consumption, transmembrane electric potential and susceptibility to calcium-induced MPTP opening were evaluated. Caspase-9 activity was measured in the intact tissue. Endurance training induced significant increases in state 3 and in respiratory control ratio both with complex I and II-linked substrates (malate+pyruvate and succinate, respectively). Increased CCCP-induced uncoupled respiration with succinate as substrate was also observed (p<0.05). No differences were found regarding state 4 and ADP/O ratio with both substrates. In addition, training significantly decreased the phosphorylative lag phase, whereas no changes were observed on maximal transmembrane electric potential, ADP-induced depolarization and repolarization potential (p<0.05). Interestingly and as opposed to our hypothesis, muscle mitochondria isolated from trained rats were more susceptible to MPTP induction by calcium, although in an initial phase muscle mitochondria isolated from trained rats had an increased calcium uptake. Interestingly, we also verified that endurance training increased the activity of caspase 9. The data obtained confirms that endurance training results in a general improvement in the gastrocnemius mitochondrial respiratory function, although mitochondrial and cellular alterations during training also result in increased calcium-induced MPTP opening.