Purpose The purpose of this study was to compare the development and etiology of neuromuscular fatigue of the knee extensor muscles at different muscle-tendon unit (MTU) lengths during repeated maximal voluntary isometric contractions (MVIC) between boys and men. Methods Twenty-two prepubertal boys (9-11 yr) and 22 men (18-30 yr) performed three knee extensor fatigue protocols at short (SHORT), optimal (OPT), and long (LONG) MTU lengths, consisting of repeating 5-s MVIC interspersed with 5-s passive recovery periods until torque reached 60% of the initial MVIC torque. The etiology of neuromuscular fatigue was identified using noninvasive methods such as surface electromyography, near-infrared spectroscopy, magnetic nerve stimulation and twitch interpolation technique. Results The number of repetitions was significantly lower in men at OPT (14.8 ± 3.2) and LONG (15.8 ± 5.8) than boys (39.7 ± 18.4 and 29.5 ± 10.2, respectively; P < 0.001), whereas no difference was found at SHORT between both age groups (boys, 33.7 ± 15.4; men, 40.9 ± 14.2). At OPT and LONG boys showed a lower reduction in the single potentiated twitch (Qtwpot) and a greater decrease in the voluntary activation level than men. At SHORT, both populations displayed a moderate Qtwpot decrement and a significant voluntary activation reduction (P < 0.001). The differences in maximal torque between boys and men were almost twice greater at OPT (223.9 N˙m) than at SHORT (123.3 N˙m) and LONG (136.5 N˙m). Conclusions The differences in neuromuscular fatigue between children and adults are dependent on MTU length. Differences in maximal torque could underpin differences in neuromuscular fatigue between children and adults at OPT and SHORT. However, at LONG these differences do not seem to be explained by differences in maximal torque. The origins of this specific effect of MTU length remain to be determined.