Free fatty acid (FFA) availability increases several-fold during exercise and remains significantly elevated for at least 3 to 6 hours after exercise cessation. Little, however, is known regarding the duration of the postexercise rise in FFA flux. In the present study, we used stable isotope-labeled palmitate infusion to examine fatty acid metabolism in 27 healthy untrained men and women (age, 29 +/- 7 years; body mass index, 25 +/- 4 kg/m²) between 13 to 16 hours and 21 to 24 hours after a single bout of moderate-intensity endurance exercise (1-2 hours at 60% of peak oxygen consumption), performed in the evening, and after a time-matched resting trial. Postabsorptive FFA rate of appearance (Ra) and FFA concentration in plasma were significantly greater after exercise than rest throughout the recovery period (P < .015), but the exercise-induced increases declined from approximately 40% at 13 to 16 hours to approximately 10% at 21 to 24 hours postexercise (P = .001). The magnitude of the exercise-induced increase in plasma FFA concentration was proportional to the increase in FFA Ra. Correlation analysis demonstrated that exercise-induced changes in plasma FFA Ra at 13 to 16 hours are (1) negatively associated with resting plasma FFA Ra and (2) positively associated with the net energy expenditure of exercise and the exercise-induced changes in whole-body fat oxidation rate (all P values < .05). In multivariate stepwise linear regression analysis, baseline plasma FFA Ra (P < or = .008) and net energy expenditure of exercise (P < or = .005) independently predicted the exercise-induced change in plasma FFA Ra at 13 to 16 hours. We conclude that the exercise-induced increase in FFA mobilization is (1) long-lived, persisting for 12 to 24 hours after exercise, with a progressive decline with time; (2) greater in subjects with low than high resting plasma FFA availability; and (3) greater after exercise with high than low energy demand.