Elevated cardiac troponin I (cTnI), a marker for cardiac damage, has been reported after high-intensity exercise in healthy subjects. Currently, little is known about the impact of prolonged moderate-intensity exercise on cTnI release, but also the impact of obesity on this response. 97 volunteers (55 men and 42 women), stratified for BMI, performed a single bout of walking exercise (30-50 km). We examined cTnI-levels before and immediately after the exercise bout in lean (BMI < 25 kg/m(2), n = 30, 57 ± 19 years), overweight (25 ≤ BMI < 30 kg/m(2), n = 29, 56 ± 11 years), and obese subjects (BMI ≥ 30 kg/m(2), n = 28, 53 ± 9 years). Walking was performed at a self-selected pace. cTnI was assessed using a high-sensitive cTnI-assay (Centaur; clinical cut-off value ≥ 0.04 μg/L). We recorded subject characteristics (body weight, blood pressure, presence of cardiovascular risk) and examined exercise intensity by recording heart rate. Mean cTnI-levels increased significantly from 0.010 ± 0.006 to 0.024 ± 0.046 μg/L (P < 0.001). The exercise-induced increase in cTnI was not different between lean, overweight and obese subjects (two-way ANOVA interaction; P = 0.27). In 11 participants, cTnI was elevated above the clinical cut-off value for myocardial infarction. Logistic regression analysis identified exercise intensity (P < 0.001), but not BMI, body fat percentage or waist circumference to significantly relate to positive troponin tests. In conclusion, prolonged, moderate-intensity exercise results in a comparable increase in cTnI-levels in lean, overweight and obese subjects. Therefore, measures of obesity unlikely relate to the magnitude of the post-exercise elevation in cTnI.