This study investigated the effect of an acute bout of exercise (>85% VO2Max) on biochemical, hemodynamic and oxidative stress variables in sedentary and physically active subjects with type 2 diabetes (T2D). Blood measurements were taken before and after a treadmill test on 12 sedentary non-diabetes subjects (ND), 12 sedentary type 2 diabetes (T2S) and 9 physically active T2D subjects (T2DA). T2DS subjects before and after the treadmill test showed a higher plasma glucose (123.2 +/- 19.0 mg/dL versus 108.9 +/- 16.8 mg/dL, p < 0.001), HbA1C (8.7 +/- 2.4% versus 7.3 +/- 1.2%, p < 0.001) and body fat% (21.3 +/- 5.7% versus 34.6 +/- 4.5%, p < 0.001) than T2DA subjects. T2DA had higher VO2Max (37.7 +/- 3.5 versus 29.5 +/- 3.2, p < 0.05), time on treadmill (22.3 +/- 2.1 min versus 16.1 +/- 2.1 min, p < 0.05), hemoglobin (17.9 +/- 0.9 g/dL, p < 0.05) and lower blood pressure levels in comparison to ND and T2DS subjects. Thiobarbituric acid substances (TBARS) in T2DS were higher than in T2DA subjects (0.27 +/- 0.1 nmol/mL versus 0.21 +/- 0.1 nmol/mL, p < 0.05). Glutathione (GSH) levels were similar among the groups. Physically active type 2 diabetes subjects had a more favorable biochemical, hemodynamic and oxidative stress profile than sedentary subjects. The coexistence of a poor cardiopulmonary performance and high oxidative stress environment can determine a profile of high risk for serious cardiovascular events in patients with diabetes.