Fine wire (FW) intramuscular electrodes and spectral analysis have not previously been used to quantify metabolic muscle fatigue in deep muscles not accessible with surface electrodes. This study compares initial median frequency (IMF) and decline in median frequency with fatigue (SLOPE) using surface and FW electrodes. Eighteen men performed isometric biceps contractions for 100 s. Electromyographic signals were collected using FW and surface electrodes. The recordings of SLOPE was greater with FW (-0.44v-0.23%IMF/s) and IMF was higher (195v69 Hz). Intrasession reliability for slope was better with FW electrode (intraclass correlation coefficient (ICC) = 0.74;P< 0.0001) than with the surface electrode (ICC = 0.43;P= 0.006), but intersession reliability was best with the surface electrode (ICC = 0.50;P= 0.03). Spectral analysis using FW electrodes provides earlier detection of muscle fatigue and can be used in deep muscles, but the reliability must be improved before clinical application.