Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (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 (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (V̇o2peak) in the T thigh was greater than that in the UT thigh ( P < 0.05); V̇o2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3 ± 3.7 vs. 1.0 ± 0.1 μmol·min−1·kg wet wt−1, P < 0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33 ± 0.07 vs. 0.02 ± 0.01 mmol/kg dry wt (dw), P < 0.05) and malate (2.2 ± 0.4 vs. 0.5 ± 0.03 mmol/kg dw, P < 0.05) and a decrease in 2-oxoglutarate (12.2 ± 1.6 vs. 32.4 ± 6.8 μmol/kg dw, 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 V̇o2peak in either trained or untrained muscle.