Oxidative stress is an important pathogenic factor of cancer and cardiovascular, metabolic and degenerative diseases. On the other hand, mild oxidative stress, as in case of physical exercise, can increase the antioxidant defense system. However, the mechanisms underlying such desirable effects of mild oxidative stress are not well understood, because the production of hydroxyl radical, the most aggressive oxygen free radical, was not yet evaluated under physiological circumstances. Therefore, in this study, we evaluated the overall production of hydroxyl radical using blood samples of ten healthy male students before and 1 h after ergometry. One h before exercise, they took salicylic acid (1g) orally so that hydroxyl radical was trapped with salicylic acid, yielding a measurable reaction product, 2,3-dihydroxybenzoic acid. Oxidative stress response to exercise was also evaluated in the volunteers without premedication by measuring serum peroxides and total antioxidant capacity of serum. These parameters of oxidative stress were then correlated with physical performance of the subjects. Ergometry caused an increase of the plasma hydroxyl radical level by 37.5% (p < 0.05), whereas the levels of total serum peroxides did not change significantly. Total serum antioxidant capacity, measured as uric acid equivalents, was higher after ergometry by 39.7% (p < 0.05), and was in positive correlation (r = 0.81) with anaerobic threshold, an indicator of physical condition. Hence, ergometry induces hydroxyl radical production and systemic oxidative stress response in the healthy subjects. Egometry could be used to study physiological oxidative stress response and to improve antioxidant defense capacities in humans.