High rates of variability in the amplitude of transcranial magnetic stimulation (TMS)-induced motor evoked potentials (MEPs), a popular method for assessing corticospinal excitability (CSE), make it essential to examine inherent reliability of the MEP amplitude. We aimed to investigate the effects of different intertrial intervals (ITIs) of single-pulse TMS on the amplitude, variability, and test-retest reliability of MEPs. Twenty-five TMS single pulses were recorded at four different ITIs of 5, 10, 15, and 20 sec from 15 healthy participants who attended two experimental sessions. Repeated measures analysis of variance (rmANOVA) and standardized z-value standard deviations (SDs) were used to investigate the effects of ITIs on MEP amplitudes and variability. Test-retest reliability of MEP amplitudes was also assessed using rmANOVA and intraclass correlation (ICC). rmANOVA revealed significantly larger MEP amplitudes following ITIs of 10, 15, and 20 sec compared with ITI 5, with no significant increases between ITIs of 15 and 20 sec. Standardized z-value SDs revealed variability rate reduction following longer ITIs with significant reductions occurring following ITIs of 10, 15, and 20 sec compared with ITI 5 with no significant difference between ITIs of 15 and 20 sec. rmANOVA showed no significant Time main effect on the MEP changes confirming within- and between-session agreement. ICCs reported significant within- and between-session reliability in all selected ITIs. The findings of the current study indicate that longer ITIs up to 15 sec can significantly induce larger MEPs with lower variability and higher reliability. The increase in ITIs not only reduces the chance of TMS-induced changes in CSE but also helps us to use this assessment tool in studies with smaller sample sizes.