Exercise therapy improves mitochondrial function in patients with mitochondrial myopathy (MM). We undertook this study to determine the metabolic abnormalities that are improved by exercise therapy. This study identified metabolic pathology using (31)P-magnetic resonance spectroscopy and magnetic resonance imaging (MRI) in a group of patients with MM compared to a control group matched for age, gender, and physical activity. We also observed the effect of exercise therapy for 12 weeks on muscle metabolism and physical function in the MM group. During muscle activity, there was impaired responsiveness of the mitochondria to changes in cytosolic adenosine diphosphate concentration, increased dependence on anaerobic energy pathways, and an adaptive increase in proton efflux in patients with MM. Following exercise therapy, mitochondrial function and muscle mass improved without any change in proton efflux rate. These metabolic findings were accompanied by improvements in functional ability. We conclude that there are significant metabolic differences between patients with MM and a control population, independent of age, gender, and physical activity. Exercise therapy can assist in improving mitochondrial function in MM patients.