Adaptation of skeletal muscle to repeated bouts of endurance exercise increases aerobic capacity and improves mitochondrial function. However, the adaptation of human skeletal muscle mitochondrial proteome to short-term endurance exercise training has not been investigated. Eight sedentary males cycled for 60min at 80% of peak oxygen consumption (VO2peak) each day for 14 consecutive days, resulting in an increase in VO2peak of 17.5±3.8% (p<0.01). Mitochondria-enriched protein fractions from skeletal muscle biopsies taken from m. vastus lateralis at baseline, and on the morning following the 7th and 14th training sessions were subjected to 2-D DIGE analysis with subsequent MS followed by database interrogation to identify the proteins of interest. Thirty-one protein spots were differentially expressed after either 7 or 14 days of training (ANOVA, p<0.05). These proteins included subunits of the electron transport chain, enzymes of the tricarboxylic acid cycle, phosphotransfer enzymes, and regulatory factors in mitochondrial protein synthesis, oxygen transport, and antioxidant capacity. Several proteins demonstrated a time course-dependent induction during training. Our results illustrate the phenomenon of skeletal muscle plasticity with the extensive remodelling of the mitochondrial proteome occurring after just 7 days of exercise training suggestive of enhanced capacity for adenosine triphosphate generation at a cellular level. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.