Blood flow occlusion (BFO) has been used to study the influence of group III/IV muscle afferents after fatiguing exercise but it is unknown how BFO-induced activity of these afferents affects motor cortical and motoneuronal excitability during low-intensity exercise. Therefore, the purpose of this study was to assess the acute effect of BFO on peripheral (maximal M-wave, M), spinal (cervicomedullary motor evoked potential, CMEP normalized to M) and motor cortical (motor evoked potential, MEP normalized to CMEP) excitability. Nine healthy males completed a sustained isometric contraction of the elbow flexors at 20% of maximal force under three conditions: i) contractile failure with BFO; ii) a time-matched trial without restriction (free flow, FFiso); iii) contractile failure with free flow (FFfail). Time-to-failure for BFO (and FFiso) was ~80% shorter than FFfail (P < 0.05). For both FFfail and FFiso, M area decreased ~17% and ~7%, respectively (P < 0.05), with no change during BFO. CMEP/M area increased ~226% and ~80% during BFO and FFfail, respectively (P < 0.05), with no change during FFiso (P > 0.05). The increase in normalized CMEP area was greater for BFO and FFfail compared with FFiso, and for BFO compared to FFfail. MEP/CMEP area was not different among the protocols (P > 0.05) and increased ~64% with time (P < 0.05). It is likely that group III/IV muscle afferent feedback to the spinal cord modulates the large increase in motoneuronal excitability for the BFO compared to FFfail and FFiso protocols.