The purpose of this investigation was to compare intramyocellular lipid (IMCL) changes in skeletal muscle in nine moderately trained subjects after 45 min of interval cycling and through 1 h of recovery. The exercise session was continuous with alternating cycling intensity achieving 50 (3 min) and 110% (2 min) of ventilatory threshold. Spectra from the vastus lateralis were acquired before, immediately after, and 60 min following exercise using a 1.5 T Signa whole-body magnet (point-resolved spectroscopy sequence, echo time 60 ms, transverse relaxation time 2000 ms, 128 acquisitions, and 20 mm3 voxel). Immediately following exercise, IMCL concentration decreased 38% compared to pre-exercise levels (P < 0.05). Fitness level and baseline IMCL were not correlated with changes in IMCL following exercise (P > 0.05). In the 60-min recovery, IMCL was reduced 30% compared to baseline (P < 0.05) and did not recover. In contrast, a nonexercising control group showed no change in IMCL. Our results suggest that IMCL decreased significantly following 45 min of interval cycling, with little recovery in the hour following.