Monitoring surface electromyographic (EMG) signals can provide useful insights for characterizing muscle fatigue, which is defined as an exercise-induced strength loss. This experiment investigated the muscle fiber conduction velocity (CV) changes induced by an endurance run. The day before and immediately after a half-marathon run (21.097 km) 11 amateur runners performed maximum voluntary contractions (MVCs) of knee extensor muscles. During the MVC, multichannel EMG was recorded from the vastus lateralis and EMG amplitude and CV were calculated. After the run, knee extensors showed a decreased strength (−13 ± 9%, p = 0.001) together with a reduction in EMG amplitude (−13 ± 10%, p = 0.003) and in CV (−6 ± 8%, p = 0.032). Knee extensor strength loss positively correlated with vastus lateralis CV differences (r = 0.76, p = 0.006). Thus, the exercises-induced muscle fatigue was associated not only with a decrease in EMG amplitude, but also with a reduction in CV. This finding suggests that muscle fibers with higher CV (i.e. those with greater fiber size) were the most impaired during strength production after an endurance run.