Aging is associated with an increase in oxidative stress‐mediated toxicity and mitochondrial dysfunction. The objectives of this study were to investigate the molecular imprint of aging on mitochondrial dysfunction and oxidative damage, and their relationship to physical activity (PA). To do this, we measured the protein content of vastus lateralis citrate synthase (CS), electron transport chain (COX subunit –II and –IV), antioxidant enzymes (Mn‐SOD and Cu/Zn‐SOD) and DNA repair enzymes (mitochondrial and nuclear OGG1), as well as CS and complex IV enzyme activity in physically active young (mean ± SD: 22 ± 2 y, n = 11) and old (AO, 70 ± 5 y, n = 10) and frail old (FO, 65 ± 10 y, n = 12) subjects. The FO subjects were patients with osteoarthritis undergoing total joint arthroplasty. The FO group had significantly lower CS and COX‐IV protein content vs. both the young (CS, P = 0.002; COX‐IV, P = 0.003) and AO (CS, P = 0.008; COX‐IV, P = 0.02) groups. COX‐II content was lower in the FO vs. the young group only (P = 0.01). Complex IV activity was lower in the FO vs. both the young (P = 0.005) and AO (P = 0.01) groups. CS activity and Mn‐SOD protein content were lower in both older groups (AO: CS activity, P = 0.05 and Mn‐SOD, P = 0.0006; FO: CS activity, P = 0.0001 and Mn‐SOD, P = 0.0001) vs. the young group. Mitochondrial OGG1 was higher in the FO vs. both the young (P=0.03) and AO (P = 0.02) groups. There was no difference in nuclear OGG1 and Cu/Zn‐SOD protein content between the three groups. These findings imply that the lower mitochondrial capacity of FO adults may be associated with lower antioxidant enzyme protection, and that PA can attenuate age‐induced mitochondrial dysregulation in the elderly population. (Funded by NSERC Canada)