Many studies have shown that the initial median frequency (MDF) and slope correlate with the muscle fiber composition. This study tested the hypothesis that the initial MDF and slope are fixed, regardless of the interval at which data are collected. MDF data using moving fast Fourier transformation of EMG signals, following local fatigue induced by isotonic exercise, were obtained. An inverse FFT was used to eliminate noise, and characteristic decreasing regression lines were obtained. The regression analysis was done in three different periods, the first one third, first half, and full period, looking at variance in the initial MDF, slope, and fatigue index. Data from surface EMG signals during fatiguing isotonic exercise of the biceps brachii and vastus lateralis in 20 normal subjects were collected. The loads tested were 30% and 60% maximum voluntary contraction (MVC) in the biceps brachii and 40% and 80% MVC in the vastus lateralis. The rate was 25 flexions per minute. There were no significant differences in the initial MDF or slope during the early or full periods of the regression, but there was a significant difference in the fatigue index. Therefore, to observe the change in the initial MDF and slope of the MDF regression line during isotonic exercise, this study suggest that only the early interval need to be observed.