Using a randomly assigned crossover design, we evaluated the change in intramyocellular lipid stores (IMCL) from baseline after a 2-h treadmill run [67% of maximal oxygen uptake (VO2 max)] and the recovery of IMCL in response to a postexercise very low-fat (10% of energy, LFAT) or moderate-fat (35% of energy, MFAT) recovery diet in seven female runners. IMCL was measured in soleus muscle by use of water-suppressed 1H-NMR spectroscopic imaging before (baseline), after, and approximately 22 h and 70 h after the run. IMCL fell by approximately 25% (P < 0.05) during the endurance run and was dependent on dietary fat content for postexercise recovery (P = 0.038, diet x time interaction). Consumption of the MFAT recovery diet allowed IMCL stores to return to baseline by 22 h and to overshoot (vs. baseline) by 70 h postexercise. In contrast, consumption of the LFAT recovery diet did not allow IMCL stores to return to baseline even by 70 h after the endurance run (P < 0.01 at 70 h). These results suggest that a certain quantity of dietary fat is required to replenish IMCL after endurance running.