The study compared the net decline of intramyocellular lipids (IMCL) during exercise (n = 18) measured by biochemical assay (BIOCH) and Oil-Red-Oil-staining (ORO) on biopsy samples from m.vastus lateralis, and by ¹H-NMR spectroscopy (MRS) sampled in a 11x11x18mm³ voxel in the same muscle. IMCL was measured before and after a 2h cycling bout (75% VO₂peak). ORO and MRS measurements showed substantial IMCL use during exercise of 31 ± 12% and 47 ± 6% of pre-exercise IMCL content. In contrast, use of BIOCH 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 BIOCH vs. MRS (r = .38, p = 0.13) or ORO (r = .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 non-invasive however not fiber type specific and hampered by an at least 30min delay in measurements after exercise completion and may overestimate IMCL use. BIOCH is the only quantitative method but is subject to variation when biopsies have different fiber type composition. However, BIOCH yields lower IMCL breakdown when compared with ORO and MRS. ORO has the advantage that it is fiber type specific and therefore provides information that is not available with other methods.