Exaggerated jaw motor activities during sleep are associated with muscle symptoms in the jaw-closing rather than the jaw-opening muscles. The intrinsic activity of antagonistic jaw muscles during sleep remains unknown. This study aims to assess the balance of muscle activity between masseter (MA) and digastric (DG) muscles during sleep in guinea pigs. Electroencephalogram (EEG), electroocculogram and electromyograms (EMGs) of dorsal neck (dNE), MA and DG muscles were recorded with video during sleep-wake cycles. These variables were quantified for each 10-second epoch. The magnitude of muscle activity during sleep in relation to mean EMG activity of total wakefulness was up to three times higher for MA muscle than for DG muscle for non-rapid eye movement (NREM) and rapid-eye-movement (REM) sleep. Although the activity level of the two jaw muscles fluctuated during sleep, the ratio of activity level for each epoch was not proportional. Epochs with a high activity level for each muscle were associated with a decrease in delta EEG power and/or an increase in heart rate in NREM sleep. However, this association with heart rate and activity levels was not observed in REM sleep. These results suggest that in guinea pigs, the magnitude of muscle activity for antagonistic jaw muscles is heterogeneously modulated during sleep, characterized by a high activity level in the jaw-closing muscle. Fluctuations in the activity are influenced by transient arousal levels in NREM sleep but, in REM sleep, the distinct controls may contribute to the fluctuation. The above intrinsic characteristics could underlie the exaggeration of jaw motor activities during sleep (e.g., sleep bruxism).