Introduction. This study aimed at comparing the rate of exercise-induced fat oxidation and postexercise free-living physical activity after constant-load flat running (FR) and downhill running (DHR) bouts at an intensity that elicited maximal fat oxidation. Methods. Participants were 11 healthy untrained men (mean age 25.6±3.3 years; VO2max39.11±8.05 ml/kg/min). The study included four visits. The first two visits determined the intensity of maximal fat oxidation during incremental FR and DHR tests. The second two visits involved constant-load FR or DHR at the intensity that elicited maximal fat oxidation in a counterbalanced order separated by two weeks. Gas exchange analysis was used to measure substrate oxidation during all exercise sessions. Sedentary time and physical activity were measured using ActiGraph triaxial accelerometers for three days including the day of exercise tests (the second day). Results. During the incremental exercise tests, fat oxidation was significantly greater during the first stage of FR (P<0.05) but started to increase during the fourth stage of DHR, although this did not reach significance. Of the 11 participants, 7 had greater fat oxidation during DHR. During continuous constant-load running, fat oxidation was higher during DHR than FR but at only two stages was either significant or borderline significant, and the time/group interaction was not significant. There was no significant effect on sedentary time of time/group interaction (P=0.769), but there was a significant effect of time (P=0.005), and there was no significant effect on total physical activity of time/group interaction (P=0.283) or time (P=0.602). Conclusion. Acute aerobic eccentric exercise at an intensity eliciting maximal fat oxidation enhanced exercise-induced fat oxidation without worsening postexercise free-living physical activity, indicating it could be a useful training modality in weight management programs.