The aim of this study was to investigate how physical fitness level could affect antioxidant activity and malondialdehyde (MDA) level at rest and in response to exhaustive exercise in healthy older adults. Fifty older adults (average age: 66.1 ± 3.8 years) were divided according to their physical fitness level into an unfit group (UG) (n = 15), a low fitness level group (LFG) (n = 18), and a high fitness level group (HFG) (n = 17). Fitness status was classified based on answers to a questionnaire about physical activity in the previous 12 months. Before and after an incremental cycle ergometer test to exhaustion, the following markers were assessed: superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase, ascorbic acid, α-tocopherol, and MDA. At rest, SOD, GPX, and α-tocopherol activities were higher in the HFG (p < 0.05), whereas MDA level was lower in the LFG in comparison with the 2 other groups (p < 0.05). During the postexercise period, antioxidant activity increased only in the LFG and the HFG (GPX, SOD, and α-tocopherol). MDA level increased in all groups after the exercise (p < 0.05). In addition, MDA level was higher during the recovery period in the HFG as compared with the others groups. This study concluded that both low and high physical fitness levels help maintain better antioxidant defenses in older adults. However, a higher physical fitness level, rather than a lower physical fitness level, could increase lipid peroxidation.