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When assessing neuromuscular fatigue (NMF) from dynamic exercise using large muscle mass (e.g., cycling), most studies have delayed measurement for 1 to 3 min after task failure. This study aimed to determine the reliability of an innovative cycling ergometer permitting the start of fatigue measurement within 1 s after cycling.Twelve subjects participated in two experimental sessions. Knee-extensor NMF was assessed by electrical nerve and transcranial magnetic stimulation with both a traditional chair setup (PRE- and POST-Chair, 2 min postexercise) and the new cycling ergometer (PRE, every 3 min during incremental exercise and POST-Bike, at task failure).The reduction in maximal voluntary contraction force POST-Bike (63% ± 12% PRE; P < 0.001) was not different between sessions and there was excellent reliability at PRE-Bike (intraclass correlation coefficient [ICC], 0.97; coefficients of variation [CV], 3.2%) and POST-Bike. Twitch (Tw) and high-frequency paired-pulse (Db100) forces decreased to 53% ± 14% and 62% ± 9% PRE, respectively (P < 0.001). Both were reliable at PRE-Bike (Tw: ICC, 0.97; CV, 5.2%; Db100: ICC, 0.90; CV, 7.3%) and POST-Bike (Tw: ICC, 0.88; CV, 11.9; Db100: ICC, 0.62; CV, 9.0%). Voluntary activation did not change during the cycling protocol (P > 0.05). Vastus lateralis and rectus femoris M-wave and motor-evoked potential areas showed fair to excellent reliability (ICC, 0.45–0.88). The reduction in maximal voluntary contraction and Db100 was greater on the cycling ergometer than the isometric chair.The innovative cycling ergometer is a reliable tool to assess NMF during and immediately postexercise. This will allow fatigue etiology during dynamic exercise with large muscle mass to be revisited in various populations and environmental conditions.