Prolonged exercise can induce cardiac troponin release. As single bouts of exercise may protect against cardiac injury, we explored the hypothesis that the magnitude of exercise-induced release of troponin attenuates upon successive days of exercise. We also examined whether effects of successive exercise bouts differ between healthy participants and individuals with cardiovascular risk factors (CVRFs) and established cardiovascular disease (CVD). We examined cardiac troponin I (cTnI) concentrations from whole venous blood samples collected from the antecubital vein (10 mL) in 383 participants (61 ± 14 yr) at rest and immediately following four consecutive days of long-distance walking (30-50 km/day). Participants were classified as either healthy (n = 222), CVRF (n = 75), or CVD (n = 86). Baseline cTnI concentrations were significantly higher in participants with CVD and CVRF compared with healthy (P < 0.001). Exercise-induced elevations in cTnI were observed in all groups following all days of walking compared with baseline (P < 0.001). Tobit regression analysis on absolute cTnI concentrations revealed a significant day × group interaction (P = 0.04). Following day 1 of walking, post hoc analysis showed that exercise-induced elevations in cTnI attenuated on subsequent days in healthy and CVRF, but not in CVD. Odds ratios for incident cTnI concentrations above the upper reference limit were significantly higher compared with baseline on day 1 for healthy participants (4.90 [95% CI, 1.58-15.2]) and participants with CVD (14.9 [1.86-125]) and remained significantly higher than baseline on all subsequent days in CVD. The magnitude of postexercise cTnI concentrations following prolonged walking exercise significantly declines upon repeated days of exercise in healthy individuals and those with CVRF, whereas this decline is not present in patients with CVD.NEW & NOTEWORTHY We show the magnitude of postexercise cardiac troponin concentrations following prolonged walking exercise significantly declines upon repeated days of exercise in healthy individuals and those with cardiovascular risk factors, while this decline is not present in patients with established cardiovascular disease.