The development of fatigue during single-joint isolated muscle contractions is accompanied by an increase in long-interval intracortical inhibition (LICI). However, the effect of whole-body locomotor endurance exercise on LICI is unknown. Eighteen healthy men completed three exercise trials on a cycle ergometer. The first trial was completed to determine the lactate threshold (LT) and maximal oxygen uptake ([Formula: see text]). The remaining two trials (familiarisation and experimental) involved cycling to volitional exhaustion at an intensity equivalent to halfway between the LT and [Formula: see text] (50%Δ). Responses to stimulation of the femoral nerve [motor nerve stimulation (MNS)] and motor cortex [transcranial magnetic stimulation (TMS)] were determined pre- and post-exercise to determine the level of peripheral fatigue [potentiated quadriceps twitch (Q)] and central fatigue [voluntary activation measured by MNS and TMS (VA and VA, respectively)]. Corticospinal excitability (motor evoked potentials) and intracortical inhibition [LICI and corticospinal silent period (SP)] were also measured from electromyography recordings on the vastus lateralis. There were exercise-induced reductions in maximal voluntary contraction torque (- 21 ± 10%), Q (- 37 ± 18%), VA (- 7 ± 7%) and VA (- 8 ± 10) (all P < 0.01). There were increases in the LICI ratio and reductions in SP duration from pre- to post-exercise (mean absolute change of 16 ± 14% and - 31 ± 28 s, respectively) (both P < 0.01). The pre- and post-exercise MEP amplitudes were not different (P = 0.86). The neural inhibitory circuits that mediate the LICI and SP became less excitable with fatigue following high-intensity exhaustive cycling, which could be important in the aetiology of central fatigue during whole-body locomotor endurance exercise.

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