Repetition suppression (RS) is evident as a weakened response to repeated stimuli after the initial response. RS has been demonstrated in motor-evoked potentials (MEPs) induced with transcranial magnetic stimulation (TMS). Here, we investigated the effect of inter-train interval (ITI) on the induction of RS of MEPs with the attempt to optimize the investigative protocols. Trains of TMS pulses, targeted to the primary motor cortex by neuronavigation, were applied at a stimulation intensity of 120% of the resting motor threshold. The stimulus trains included either four or twenty pulses with an inter-stimulus interval (ISI) of 1 s. The ITI was here defined as the interval between the last pulse in a train and the first pulse in the next train; the ITIs used here were 1, 3, 4, 6, 7, 12, and 17 s. RS was observed with all ITIs except with the ITI of 1 s, in which the ITI was equal to ISI. RS was more pronounced with longer ITIs. Shorter ITIs may not allow sufficient time for a return to baseline. RS may reflect a startle-like response to the first pulse of a train followed by habituation. Longer ITIs may allow more recovery time and in turn demonstrate greater RS. Our results indicate that RS can be studied with confidence at relatively short ITIs of 6 s and above.