The main aim of this study was to assess neuromuscular fatigue during a typical high-load, low-repetition loading protocol. Muscle stimulations were used to assess maximum voluntary contraction, resting single- and double-pulse twitch characteristics, and superimposed double-pulse twitch force (used to calculate voluntary activation) before and after an acute knee extension loading protocol. In our participants, who had previous resistance training experience, the mean voluntary activation level was 96.2% in an unfatigued state. Maximum voluntary contraction (-11.8%), resting double-pulse twitch force (-10.6%), and voluntary activation (-2.1%) were markedly decreased as a consequence of loading (P < 0.05). In addition, although potentiated twitch characteristics were observed during the loading protocol, this was short-lived, as fatigue surpassed the potentiation mechanisms. Our results show that both central and peripheral mechanisms contributed to neuromuscular fatigue during the present loading protocol.