Mitochondrial free radical formation has been implicated as a potential mechanism underlying degenerative senescence, although human data are lacking. Therefore, the present study was designed to examine if resting and exercise-induced intramuscular free radical-mediated lipid peroxidation is indeed increased across the spectrum of sedentary aging. Biopsies were obtained from the vastus lateralis in six young (26 ± 6 yr) and six aged (71 ± 6 yr) sedentary males at rest and after maximal knee extensor exercise. Aged tissue exhibited greater ( P < 0.05 vs. the young group) electron paramagnetic resonance signal intensity of the mitochondrial ubisemiquinone radical both at rest (+138 ± 62%) and during exercise (+143 ± 40%), and this was further complemented by a greater increase in α-phenyl-tert-butylnitrone adducts identified as a combination of lipid-derived alkoxyl-alkyl radicals (+295 ± 96% and +298 ± 120%). Lipid hydroperoxides were also elevated at rest (0.190 ± 0.169 vs. 0.148 ± 0.071 nmol/mg total protein) and during exercise (0.567 ± 0.259 vs. 0.320 ± 0.263 nmol/mg total protein) despite a more marked depletion of ascorbate and uptake of α/β-carotene, retinol, and lycopene ( P < 0.05 vs. the young group). The impact of senescence was especially apparent when oxidative stress biomarkers were expressed relative to the age-related decline in mitochondrial volume density and absolute power output at maximal exercise. In conclusion, these findings confirm that intramuscular free radical-mediated lipid peroxidation is elevated at rest and during acute exercise in aged humans.