The hypothesis that fatigue during prolonged exercise arises from insufficient intramuscular glycogen, which limits tricarboxylic acid cycle (TCA) activity due to reduced TCA cycle intermediates (TCAI), was tested in this experiment. Seven endurance-trained men cycled at ∼70% of peak O2uptake (V˙o 2 peak) until exhaustion with low (LG) or high (HG) preexercise intramuscular glycogen content. Muscle glycogen content was lower ( P < 0.05) at fatigue than at rest in both trials. However, the increase in the sum of four measured TCAI (>70% of the total TCAI pool) from rest to 15 min of exercise was not different between trials, and TCAI content was similar after 103 ± 15 min of exercise (2.62 ± 0.31 and 2.59 ± 0.28 mmol/kg dry wt for LG and HG, respectively), which was the point of volitional fatigue during LG. Subjects cycled for an additional 52 ± 9 min during HG, and although glycogen was markedly reduced ( P < 0.05) during this period, no further change in the TCAI pool was observed, thus demonstrating a clear dissociation between exercise duration and the size of the TCAI pool. Neither the total adenine nucleotide pool (TAN = ATP + ADP + AMP) nor IMP was altered compared with rest in either trial, whereas creatine phosphate levels were not different when values measured at fatigue were compared with those measured after 15 min of exercise. These data demonstrate that altered glycogen availability neither compromises TCAI pool expansion nor affects the TAN pool or creatine phosphate or IMP content during prolonged exercise to fatigue. Therefore, our data do not support the concept that a decrease in muscle TCAI during prolonged exercise in humans compromises aerobic energy provision or is the cause of fatigue.