Human studies have not assessed supraspinal or spinal motoneurone excitability in the quiescent state prior to a rhythmic and alternating cyclical motor output. The purpose of the current study was to determine whether supraspinal and (or) spinal motoneurone excitability was modulated in humans prior to arm cycling when compared with rest with no intention to move. We hypothesized that corticospinal excitability would be enhanced prior to arm cycling due, in part, to increased spinal motoneurone excitability. Supraspinal and spinal motoneurone excitability were assessed via transcranial magnetic stimulation (TMS) of the motor cortex and transmastoid stimulation of the corticospinal tract, respectively. Surface electromyography recordings of TMS motor evoked potentials (MEPs) and cervicomedullary MEPs (CMEPs) were made from the relaxed biceps brachii muscle prior to rhythmic arm cycling and at rest with no intention to move. The amplitude of the MEPs was greater (mean increase: +9.8% of maximal M wave; p = 0.006) and their onset latencies were shorter (mean decrease: -1.5 ms; p < 0.05) prior to cycling when compared with rest. The amplitudes of the CMEPs at any of 3 stimulation intensities were not different between conditions. We conclude that premovement enhancement of corticospinal excitability is greater prior to arm cycling than at rest because of increases in supraspinal but not spinal motoneurone excitability.