To assess oxidative stress (OS) induced by endurance exercise, concentrations of serum reactive oxygen species (ROS) were determined in 70 Japanese male amateur runners completing a two-day ultra-marathon race. Serum ROS levels were analyzed at three time points: before the race (baseline), after the 1st day race (mid-race), and after the 2nd day race (goal) (post-race). The means (SE) of ROS were 151.4(3.7) (U. CARR.), 168.7(4.4), and 156.8(4.4), respectively. Significant positive trends were noted between age and serum ROS concentrations at the three race points (p<0.05 for all). After adjusting for age, BMI and average monthly running distance, the baseline serum ROS concentrations were positively associated with completion times of the first-day race, in particular (p<0.05), suggesting that the concentrations may predict physical performance. The ROS production increased at mid-race (p<0.05), but the levels returned to baseline levels at post-race, indicating that an antioxidant defense system may develop post-race to reduce OS.