It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H₂O₂ emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H₂O₂ emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H₂O₂ and the fraction of electron leak to H₂O₂, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H₂O₂ emission rates in the presence of ADP, the fraction of electron leak to H₂O₂, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H₂O₂ emission and contributes to age-associated redox stress.