Single bouts of exercise induce an acute state of oxidative stress. It is largely unknown what this means in the context of diseases which are associated with increased oxidative stress, e.g., type 2 diabetes mellitus (T2DM). Free radicals can destroy the structure of erythrocytes and reduce their deformability. Antioxidative peroxiredoxins are highly abundant in erythrocytes. Therefore, we immunohistochemically examined whether the free radical-induced erythrocyte lipid-peroxidation measured by 8-iso-prostaglandin-F2α (8-Iso-PGF) as well as the erythrocyte contents of overoxidized peroxiredoxins (PRDX-SO(2-3)) differ between overweight/obese T2DM men (n = 15, years = 59 ± 10 (mean ± SD)) and overweight/obese non-diabetic control subjects (n = 12, years = 53 ± 4) during acute exercise (WHO-step test). We further studied whether physical training affects the oxidative stress response to acute exercise. Seven men belonging to the diabetic group took part in a moderate intensity cycling endurance training. Erythrocyte 8-Iso-PGF significantly increased during acute exercise and decreased in the 30-min recovery phase in untrained diabetic and non-diabetic men (P ≤ 0.05). Increases/decreases in 8-Iso-PGF in relation to exercise/recovery time were similar in both groups. A significant exercise-induced increase in the contents of erythrocyte PRDX-SO(2-3) was only observed in T2DM men (P ≤ 0.05). PRDX-SO(2-3) contents were not reduced during recovery. Following physical training, the magnitude of exercise-induced increases in 8-Iso-PGF (relative to exercise time) was significantly lower in the erythrocytes of T2DM men (P ≤ 0.05), whereas increases in PRDX-SO(2-3) were significantly higher (P ≤ 0.05). Exercise-induced erythrocyte lipid-peroxidation is similar in untrained overweight/obese T2DM patients and overweight/obese control subjects, while antioxidative mechanisms differ. Physical training might improve oxidative stress in T2DM men's erythrocytes during acute exercise.