Electromyographic (EMG) assessment has been used as a non-invasive tool to objectively assess muscle function, although with controversial research and clinical potential. The aim of this study was to assess within-, inter-subject and between-day repeatability of serial EMG recordings. The study sample included 10 asymptomatic subjects with no history of temporomandibular disorders or muscle parafunctions. Bilateral masseter and anterior temporalis muscle EMG parameters were assessed in two standardized serial recordings (day 1 to day 2) using a portable EMG equipment (ME 6000 recorder, Mega Electronics, Kuopio, Finland). The functional tasks included postural/resting activities as pre- and post-recording series of 30 s each and jaw opening/closing, intercuspal and maximal voluntary clenching activities of 5 s, repeated three times. The assessed EMG parameters included the mean amplitude, s.d. and error. In addition, the power spectrum EMG parameter assessment included the median power frequencies and the averaged EMG spectrum data values. The results of the intraclass correlation coefficient analysis indicated reliability for nearly all of the intercuspal and all clenching EMG amplitude and power spectrum parameters. This was complemented by the repeated measures anova and post hoc analyses that indicated non-significant differences between day 1 and 2 in task- and muscle-related analyses. Most variability was noted in postural and some in opening/closing tasks. In conclusion this study assessed the reliability, repeatability and limitations of postural and various dynamic masseter and temporalis EMG recordings for serial assessment.