Masticatory muscle thickness provides objective measurements of the oral motor function, which may change in patients with oral myofascial pain. In this study, we aimed to establish a reliable ultrasound (US) protocol for imaging the superficial and deep masticatory muscles and to identify the potential influencers of the measurements. Forty-eight healthy participants without orofacial pain were enrolled. The intra-and inter-rater reliabilities of US measurements for masseter, temporalis, and lateral pterygoid muscles were assessed. Intraclass correlation coefficients for all muscles were greater than 0.6. The generalised estimating equation was used to analyse the impact of age, gender, laterality, and body mass index on the measurements, whereby age and body mass index were likely to be associated with an increase in masticatory muscle thickness. The thickness tended to be lesser in females. Laterality seemed to exert minimal influence on masticatory muscle thickness. Our study shows acceptable reliability of US in the evaluation of superficial and deep masticatory muscle thickness. Future studies are warranted to validate the usefulness of US imaging in patients with oral myofascial pain syndrome.