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. Temporomandibular disorder (TMD) is frequently observed in patients seeking dental treatment. It comprises various findings concerning the stomatognathic system, involving the temporomandibular joints, masticatory muscles, teeth, and ears 1,2. The prevalence of TMDs in the general population ranges from 34.9 to 42.4% across different studies 3,4 , and the incidence is more prevalent in females 5. Of note, oral myofascial pain is commonplace in about 10-15% of patients with TMD 6. The aetiology of oral myofascial pain is still controversial. The proposed mechanisms include overuse of the masticatory muscles and a decrease in its pain threshold following central sensitization 7. While overuse might lead to hypertrophy of the masticatory muscles in the early stages, persistent pain could result in disuse atrophy in chronic cases 8. In this sense, masticatory muscle thickness provides objective measurements of the oral motor function 9 , which is supposed to change in patients suffering from oral myofascial pain. Several imaging modalities, such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US) have been used to image the masticatory muscles. CT allows precise delineation of the skull bone where the masticatory muscles attach. However, radiation and poor resolution of the soft tissues make it unsuitable for muscle thickness measurements. Nowadays, MRI serves as the gold standard for depicting soft tissue pathologies 10 , but it is limited by its high cost and contraindications in patients with metal implants. US has various advantages over CT and MRI, i.e. real-time evaluation, zero radiation, cost-effectiveness, portability, and ease of dynamic examination 11. Further, it is capable of delineating muscle fibres-making it highly effective for imaging muscle trauma and neuromuscular diseas...