The present study aimed to investigate the effects of a standardized fatiguing protocol on central and peripheral fatigue in knee-flexors and knee-extensors. Thirteen healthy men (age: 23 ± 3 years; height: 1.78 ± 0.09 m; body-mass: 73.6 ± 9.2 kg) volunteered for the present study. Maximal voluntary contraction (MVC), Electromyography (EMG) activity, voluntary activation level (VAL) as an index of central fatigue and twitch potentiation as an index of peripheral fatigue were measured before and after the fatiguing protocol. The fatiguing protocol consisted of a 0.6 duty-cycle to exhaustion (6 s isometric contraction, 4 s recovery) at 70% MVC. After the fatiguing protocol, MVC decreased in both (Effect-size (ES) = 1.14) and knee-extensors (ES = 1.14), and EMG activity increased in both knee-flexors (ES = 2.33) and knee-extensors (ES = 1.54). Decreases in VAL occurred in knee-flexors (ES = 0.92) but not in knee-extensors (ES = 0.04). Decreases in potentiation occurred in both knee-flexors (ES = 0.84) and knee-extensors (ES = 0.58). The greater central occurrence of fatigue in knee-flexors than in knee-extensors may depend on the different muscle morphology and coupled with a greater tolerance to fatigue in knee-extensors. The present data add further insight to the complicated knee-flexors-to-knee-extensors strength relationship and the mechanisms behind the different occurrence of fatigue.