Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Heart and skeletal muscles have a high density of mitochondria with robust energetic demands and mitochondria plasticity has an important role in both cardiovascular system and skeletal muscle responses. The aim of this study was to investigate the influence of regular physical activity on oxidation profiles of mitochondrial proteins from heart and tibialis anterior muscles. To this end, we used mouse as animal model. Mice were divided in two groups: untrained and regularly trained. The carbonylated protein pattern was studied by two-dimensional gel electrophoresis followed by Western Blot with anti-dinitrophenyl hydrazone antibodies. Mass spectrometry analysis allowed the identifications of several different protein oxidation sites including methionine, cysteine, proline and leucine residues. A large number of oxidized protein were found in both untrained and trained animals. Moreover, mitochondria from skeletal muscles and heart showed almost the same carbonylation pattern. Interestingly, exercise training seems to increase carbonylation level mostly of mitochondrial protein from skeletal muscle.