Objective: Previous research has suggested that transcranial magnetic stimulation (TMS) related cortical excitability measures could be estimated quickly using stimulus–response curves with short interstimulus intervals (ISIs). Here we evaluated the resting motor threshold (rMT) estimated with these curves. Methods: Stimulus-response curves were measured with three ISIs: 1.2–2 s, 2–3 s, and 3–4 s. Each curve was formed with 108 stimuli using stimulation intensities ranging from 0.75 to 1.25 times the rMTguess, which was estimated based on motor evoked potential (MEP) amplitudes of three scout responses. Results: The ISI did not affect the rMT estimated from the curves (F = 0.235, p = 0.683) or single-trial MEP amplitudes at the group level (F = 0.90, p = 0.405), but a significant subject by ISI interaction (F = 3.64; p < 0.001) was detected in MEP amplitudes. No trend was observed which ISI was most excitable, as it varied between subjects. Conclusions: At the group level, the stimulus–response curves are unaffected by the short ISI. At the individual level, these curves are highly affected by the ISI. Significance Estimating rMT using stimulus–response curves with short ISIs impacts the rMT estimate and should be avoided in clinical and research TMS applications.