Ageing, a sedentary lifestyle, and obesity are associated with increased oxidative stress, while regular exercise is associated with an increased antioxidant capacity in trained skeletal muscles. Whether a higher aerobic fitness is associated with increased expression of antioxidant enzymes and their regulatory factors in skeletal muscle remains unknown. Although oestrogens could promote a higher antioxidant capacity in females, it remains unknown whether a sex dimorphism exists in humans regarding the antioxidant capacity of skeletal muscle. Thus, the aim was to determine the protein expression levels of the antioxidant enzymes SOD1, SOD2, catalase and glutathione reductase (GR) and their regulatory factors Nrf2 and Keap1 in 189 volunteers (120 males and 69 females) to establish whether sex differences exist and how age, VO₂max and adiposity influence these. For this purpose, vastus lateralis muscle biopsies were obtained in all participants under resting and unstressed conditions. No significant sex differences in Nrf2, Keap1, SOD1, SOD2, catalase and GR protein expression levels were observed after accounting for VO₂max, age and adiposity differences. Multiple regression analysis indicates that the VO₂max in mL.kg LLM⁻¹.min⁻¹can be predicted from the levels of SOD2, Total Nrf2 and Keap1 (R = 0.58, P < 0.001), with SOD2 being the main predictor explaining 28 % of variance in VO₂max, while Nrf2 and Keap1 explained each around 3 % of the variance. SOD1 protein expression increased with ageing in the whole group after accounting for differences in VO₂max and body fat percentage. Overweight and obesity were associated with increased pSer⁴⁰-Nrf2, pSer⁴⁰-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels after accounting for differences in age and VO₂max. Overall, at the population level, higher aerobic fitness is associated with increased basal expression of muscle antioxidant enzymes, which may explain some of the benefits of regular exercise.