The present study was designed to test the accuracy, validity, reliability and sensitivity of the main outcomes of alternative methods for the measure of TMS-assessed voluntary activation (VATMS) in the knee extensors. Ten healthy recreationally active males (24 ± 5 years) completed a neuromuscular assessment protocol (NMA) before and immediately after a fatiguing isometric exercise, consisting of two sets of five contractions (50%, 62.5%, 75%, 87.5%, and 100% of Maximal Voluntary Contraction; MVC) with superimposed TMS-evoked twitches (SITs) for calculation of VATMS (1x5C vs. 2x5C). The protocol was performed on two separate occasions for the measurement of between-day reliability. Where deemed appropriate, comparisons were made with a routinely used protocol [i.e. 50%, 75%, and 100% of MVC (1x3C) performed three times (3x3C)] from re-analysed data (Dekerle et al., 2018). Confidence intervals for the measure of a key determinant of VATMS (estimated resting twitch) were similar between 1x5C and 2x5C but improved by six-fold when compared to 1x3C (P<0.05). Potentiated twitch force evoked via percutaneous electrical stimulation of the femoral nerve was unchanged from pre- to post-NMA at baseline for 1x5C (P>0.05) but decreased for 2x5C and 3x3C (P<0.05). Its recovery post-exercise was lesser for 1x5C compared to 2x5C and 3x3C (P<0.05), with no difference between the latter two (P>0.05). Absolute reliability was strong enough for both 1x5C and 2x5C to depict a true detectable change in the sample’s VATMS following the fatiguing exercise (TEM < 3% at rest, <9% post-exercise) but 2x5C was marginally more sensitive to individual’s changes at baseline. In conclusion, both 1x5C and 2x5C provide reliable measures of VATMS. However, the 1x5C protocol may hold stronger internal validity.