Purpose - 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. Methods - 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). Results - 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. Conclusions - 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.