Intramuscular triglycerides (IMTG) are assumed to form an important substrate source during prolonged endurance exercise in trained males. This study investigated the effects of endurance exercise and recovery diet on IMTG content in vastus lateralis muscle. Nine male cyclists were provided with a standardized diet for 3 days, after which they performed a 3-h exercise trial at a 55% maximum workload. Before and immediately after exercise and after 24 and 48 h of recovery, magnetic resonance spectroscopy (MRS) was performed to quantitate IMTG content. Muscle biopsies were taken after 48 h of recovery to determine IMTG content by using quantitative fluorescence microscopy. The entire procedure was performed two times; in one trial, a normal diet containing 39% energy (En%) as fat was provided (NF) and in the other a typical carbohydrate-rich athlete's diet (LF: 24 En% fat) was provided. During exercise, IMTG content decreased by 21.4 +/- 3.1%. During recovery, IMTG content increased significantly in the NF trial only, reaching preexercise levels within 48 h. In accord with MRS, fluorescence microscopy showed significantly higher IMTG content in the NF compared with the LF trial, with differences restricted to the type I muscle fibers (2.1 +/- 0.2 vs. 1.4 +/- 0.2% area lipid staining, respectively). In conclusion, IMTG content in the vastus lateralis muscle declines significantly during prolonged endurance exercise in male cyclists. When a normal diet is used, IMTG contents are subsequently repleted within 48 h of postexercise recovery. In contrast, IMTG repletion is impaired substantially when a typical, carbohydrate-rich athlete's diet is used. Data obtained by quantitative fluorescence microscopy correspond well with MRS results, implying that both are valid methods to quantify IMTG content.