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• The use of neuronavigated TMS should lead to superior targeting and stabilization of stimulus delivery. • The use of neuronavigated TMS does not influence the corticospinal responses in unfatigued and fatigued muscle, relative to a standardized function-guided procedure. • Function-guided procedure seems as accurate as neuronavigated TMS in the assessment of corticospinal excitability and inhibition in unfatigued and fatigued muscle, providing a trained investigator can reliably position and hold the TMS coil. In studying neuromuscular fatigability, researchers commonly use functional criteria to position and hold the transcranial magnetic stimulation (TMS) coil during testing sessions. This could influence the magnitude of corticospinal excitability and inhibition responses due to imprecise and unsteady positions of the coil. To reduce coil position and orientation variability, neuronavigated TMS (nTMS) could be used. We evaluated the accuracy of nTMS and a standardized function-guided procedure for maintaining TMS coil position both in unfatigued and fatigued knee extensors. Eighteen participants (10F/8M) volunteered in two identical and randomized sessions. Maximal and submaximal neuromuscular evaluations were performed with TMS three times before (PRE_1) and three times after (PRE_2) a 2 min resting session and one time immediately after (POST) a 2-min sustained maximal voluntary isometric contraction (MVIC). The located "hotspot" [the location that evoked the largest motor-evoked potential (MEP) responses in the rectus femoris ] was maintained either with or without nTMS. MEP, silent period (SP) and the distance between the "hotspot" and the actual coil position were recorded. A time × contraction intensity × testing session × muscle interaction was not observed for MEP, SP, and distance. Bland-Altman plots presented adequate agreements for MEP and SP. Spatial accuracy of TMS coil position over the motor cortex did not influence corticospinal excitability and inhibition in unfatigued and fatigued knee extensors. The variability in MEP and SP responses may be due to spontaneous fluctuations in corticospinal excitability and inhibition, and it is not altered by the spatial stability of the stimulation point. [ABSTRACT FROM AUTHOR]