Muscle glutamate is central to reactions producing 2-oxoglutarate, a TCA cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops ~40-80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e. fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1h/day at 70% maximum workload for 5 days/week), while their contralateral quadriceps remained untrained (UT). Following 5 weeks of training, VO_2peak in the T thigh was greater than UT thigh under control conditions (p<0.05); VO_2peak was not different between the T and UT thigh with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared to rest (7.3±3.7 vs 1.0±0.1 µmol/min/kg-wet-wt, p<0.05) without increasing TCA cycle intermediates. In the UT thigh, peak exercise (versus rest) induced an increase in fumarate (0.33±0.07 vs 0.02±0.01 mmol/kgdw, p<0.05) and malate (2.2±0.4 vs 0.5±0.03 mmol/kgdw, p<0.05) and decrease in 2-oxoglutarate (12.2±1.6 vs 32.4±6.8 µmol/kgdw, p<0.05). Overall, glutamate infusion increased arterial glutamate (p<0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate (p<0.05) and decreased 2-oxoglutarate (p<0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect VO_2peak in either trained or untrained muscle.