Contraction intensity is a key factor determining the development of muscle fatigue and it has been shown to induce distinct changes along the motor pathway. The role of cortical and spinal inputs that regulate motor unit (MU) behaviour during fatiguing contractions is poorly understood. We studied the cortical, spinal, and neuromuscular response to sustained fatiguing isometric tasks performed at 20 and 70% of the maximum isometric voluntary contraction (MVC), together with MUs behaviour of knee extensors in healthy active males. Neuromuscular function was assessed before and after performing both tasks. Cortical and spinal responses during exercise were measured via stimulation of the motor cortex and spinal cord. High density electromyography was used to record individual MUs from the vastus lateralis (VL). Exercise at 70% MVC induced greater decline in MVC (p = 0.023), and potentiated twitch force compared to 20%MVC (p < .001), with no difference in voluntary activation (p = 0.514). Throughout exercise, corticospinal responses were greater during the 20%MVC task (p < 0.001), and spinal responses increased over time in both tasks (p ≤ 0.042). MU discharge rate increased similarly following both tasks (p ≤ 0.043) while recruitment and de-recruitment thresholds were unaffected (p ≥ 0.295). These results suggest that increased excitability of cortical and spinal inputs might be responsible for the increase in MU discharge rate. The increase in evoked responses together with the higher MUs discharge rate might be required to compensate for peripheral adjustments to sustain fatiguing contractions at different intensities.