This study aimed to compare quadriceps function (i.e. strength, endurance, central, and peripheral fatigue) of young (Young-UnTr) and middle-aged (MidAge-UnTr) untrained men and young endurance-trained men (Young-Tr). Twenty-four male subjects (eight Young-UnTr (26 ± 4 yr), eight Young-Tr (29 ± 3 yr), and eight MidAge-UnTr (56 ± 4 yr) performed a maximal cycling test to assess their fitness level. On a separate visit, subjects performed sets of 10 intermittent (5-s on/5-s off) isometric contractions starting at 10% maximum voluntary contraction (MVC), with 10% MVC increments from one set to another until exhaustion. Electrophysiological and mechanical (e.g. twitch) evoked responses elicited with magnetic femoral nerve stimulation in the relaxed muscle and during MVC (i.e. estimation of voluntary activation using the interpolated twitch technique) were measured at baseline and after each set to assess peripheral and central fatigue, respectively. Endurance (= total number of contractions) was also evaluated. Young-UnTr exhibited larger reductions in evoked quadriceps mechanical responses than MidAge-UnTr and Young-Tr after identical standardized muscle loading (e.g. after the 50% MVC set, reduction in single potentiated twitch was -36 ± 9%, -21±16%, and -2 ± 4%, respectively). At both 50% MVC set and exhaustion, MidAge-UnTr exhibited similar reduction in maximal voluntary activation and displayed similar endurance compared to Young-UnTr. Young-Tr exhibited greater endurance than Young-UnTr without significant changes in maximal voluntary activation throughout the test. This study provides robust comparative data regarding the influence of chronic exposure to endurance training and middle-aged on central and peripheral quadriceps fatigability and endurance. Endurance-trained subjects showed smaller level of peripheral fatigue and displayed no significant central fatigue, even at exhaustion and despite greater endurance performance. Our findings also demonstrate that men in the sixth decade exhibit significant alterations in quadriceps function typically observed in much older subjects. These data emphasize the need for developing normative data for both central and peripheral quadriceps fatigability.