A new fatigue index for the assessment of peripheral muscle fatigue during isotonic contraction was defined in this study. Twenty subjects performed five sets of ten isotonic contractions with two minutes of rest between each set. Surface electromyography (sEMG) was recorded from the rectus femoris muscle for each subject. The new fatigue index ($FI_{hlrx}$) was defined as the ratio of the high frequency component to the low frequency component of raw EMG signals. The accuracy of the proposed index was compared with that of well-known EMG-based fatigue indices such as the root mean square (RMS) and median frequency ($F_{med}$). Results showed that the indices proposed in this study were more accurate than those traditional approaches. In particular, $FI_{hlr360}$ was the most strongly correlated with mechanical joint power (r = 0.749). The new fatigue index could serve as a reliable tool for the assessment of peripheral muscle fatigue, irrespective of EMG signal variability caused by dynamic contractions.