['Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development.', 'The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients.', 'We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index.', 'One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients.', 'Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training.', 'Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of β-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training.'] Is physical activity the key determinant of skeletal muscle mitochondrial function in type 2 diabetes?