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The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WRMAX) and 3 min at 100% WRMAX. EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone [0.167±0.055 vs. rest: 0.106±0.047 arbitrary units (AU)/g total protein (TP), P< 0.05] and α-phenyl-tert-butylnitrone-adducts (112±41 vs. rest: 29±9 AU/mg tissue mass, P< 0.05). Intramuscular lipid hydroperoxides also increased (0.320±0.263 vs. rest: 0.148±0.071 nmol/mg TP, P< 0.05) despite an uptake of α-tocopherol, α-carotene and β-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct …