Mitochondrial dysfunction, defined as increased oxidative stress and lower capacity for energy production, may be seen with aging and may cause frailty, or it could be that it is secondary to physical inactivity. We studied the effect of two weeks of one-leg immobilization followed by six weeks of supervised cycle training on mitochondrial function in 17 young (23 ± 1 years, mean ± SEM) and 15 older (68 ± 1 years) healthy men. Submaximal hydrogen peroxide H₂O₂ emission and respiration were measured simultaneously at a predefined membrane potential in isolated mitochondria from skeletal muscle using two protocols pyruvate+malate (PM) and succinate+rotenone (SR). This allowed measurement of leak and ATP generating respiration from which the coupling efficiency can be calculated. Protein content of the antioxidants manganese superoxide dismuthase (MnSOD), CuZn-superoxide dismuthase (CuZnSOD), catalase and gluthathione peroxidase 1 (GPX1) were measured by Western Blotting. Immobilization decreased ATP generating respiration using PM and increased H₂O₂ emission using both PM and SR similarly in young and older men. Both were restored to baseline after the training period. Furthermore, MnSOD and catalase content increased with endurance training. The young men had a higher leak respiration at inclusion using PM and a higher membrane potential in state 3 using both substrate combinations. Collectively, this study supports the notion that increased mitochondrial ROS mediates the detrimental effects seen after physical inactivity. Age on the other hand was not associated with impairments in antioxidant protein levels, mitochondrial respiration or H₂O₂ emission using either protocol. This article is protected by copyright. All rights reserved.