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Background: Strength-Duration (S-D) assessment is commonly used in clinics to examine the excitability of peripheral nerves and muscles. Yet, how changes in neuromuscular excitability relates to improved athletic and muscular performance in healthy subjects remains poorly understood. Therefore, the aim of the study was to evaluate the electrophysiological changes in neuromuscular excitability in the vastus medialis (VM) muscle, using the S-D assessment, following a back squat conditioning activity (BS-CA) protocol designed to elicit a post-activation potentiation (PAP)/post-activation performance enhancement (PAPE) effect. Methods: Eleven athletic males were included in this study. All subjects performed two trials: one examining their BS one-repetition maximum (1 RM) and a main experiment. During the main experiment, baseline levels of rectangular rheobase (R-RIC), triangular rheobase (R-DIC) and chronaxie was collected from the VM muscle, following a standard warmup. Subsequently, the subjects performed three warmup BS-sets and executed a top set of five repetitions (reps) at 80% of 1RM. Afterwards, R-RIC, R-DIC and chronaxie was reassessed for pre and post analysis. Based on these S-D curve (SDC) parameters, the muscle adjustability quotient (MAQ) and threshold charge (Q) was also computed and compared. Results: The R-RIC, R-DIC and Q were all significantly higher following the BS-CA, compared to pre-intervention (p < 0.001). No significant differences were observed for the chronaxie and MAQ (p > 0.05), although an increasing trend was noted (p = 0.054). Conclusions: Based on the findings from this study, the neuromuscular excitability in the VM muscle can be acutely altered following a BS-CA-protocol. However, these changes seems to be more related to muscle fatigue than PAP/PAPE. Nevertheless, S-D assessment may broaden our understanding of the fatigue process during exercise.