Background: This study aimed to determine whether oxidative stress was related to cardiovascular risk indices in children, and whether an exercise intervention would reduce oxidative stress. Methods: A randomized trial of two different doses of exercise and a no-exercise control group included 112 overweight and obese children, 7-11 years old. Plasma isoprostane levels were obtained at baseline and after the intervention. Cross-sectional analysis of oxidative stress and metabolic markers at baseline was performed. The effect of the exercise training on oxidative stress was tested. Results: Lower isoprostane levels were observed in blacks. At baseline, isoprostane was positively related to measures of fatness (BMI, waist circumference, percent body fat), insulin resistance and β-cell function (fasting insulin, insulin area under the curve, Matsuda index, disposition index, oral disposition index), and several lipid markers (low-density lipoprotein, triglycerides, total cholesterol), and inversely with fitness [peak oxygen consumption (VO2)], independent of race, sex, and cohort. No relation was found with visceral fat, blood pressure, or glycemia. Independent of percent body fat, isoprostane predicted triglycerides, β=0.23, total cholesterol-to-high-density lipoprotein (TC/HDL) ratio, β=0.23, and insulin resistance (insulin area under the curve, β=0.24, Matsuda index, β=-0.21, oral disposition index, β=0.33). Exercise did not reduce oxidative stress levels, despite reduced fatness and improved fitness in these children. Conclusions: Isoprostane levels were related to several markers of cardiovascular risk at baseline; however, despite reduced fatness and improved fitness, no effect of exercise was observed on isoprostane levels. To our knowledge, this is the first report in children to demonstrate a correlation of oxidative stress with disposition index, fitness, and TC/HDL ratio, the first to test the effect on oxidative stress of an exercise intervention that reduced body fat, and the first such exercise intervention study to include a substantial proportion of black children. © Copyright 2013, Mary Ann Liebert, Inc.