The study compared the net decline of intramyocellular lipids (IMCL) during exercise ( n = 18) measured by biochemical assay (BIO) and Oil Red O (ORO) staining on biopsy samples from vastus lateralis muscle and by¹H-MR spectroscopy (MRS) sampled in an 11 × 11 × 18-mm³voxel in the same muscle. IMCL was measured before and after a 2-h cycling bout (∼75% V̇o_{2 peak}). ORO and MRS measurements showed substantial IMCL use during exercise of 31 ± 12 and 47 ± 6% of preexercise IMCL content. In contrast, use of BIO for IMCL determination did not reveal an exercise-induced breakdown of IMCL (2 ± 9%, P = 0.29) in young healthy males. Correlations between different measures of exercise-induced IMCL degradation were low. Coefficients were 0.48 for MRS vs. ORO ( P = 0.07) and were even lower for BIO vs. MRS ( r = 0.38, P = 0.13) or ORO ( r = 0.08, P = 0.78). This study demonstrates that different methods to measure IMCL in human muscles can result in different conclusions with regard to exercise-induced IMCL changes. MRS has the advantage that it is noninvasive, however, not fiber type specific and hampered by an at least 30-min delay in measurements after exercise completion and may overestimate IMCL use. BIO is the only quantitative method but is subject to variation when biopsies have different fiber type composition. However, BIO yields lower IMCL breakdown compared with ORO and MRS. ORO has the major advantage that it is fiber type specific, and it therefore provides information that is not available with the other methods.