BackgroundThis study aimed to prospectively investigate the reference values for masseter muscle thickness and hardness using ultrasonography and shear wave elastography, respectively, in patients with hemifacial microsomia (HFM). MethodsWe enrolled 51 patients with HFM aged 5–20 years, including 31 males and 20 females. The upper-lower, left-right, and anterior-posterior diameters of 102 masseter muscles, and the stiffness of 98 masseter muscles were determined by examining the healthy and affected sides of each participant’s face. ResultsThe upper-lower, left-right, and anterior-posterior diameters of the masseter muscle were significantly smaller at rest (4.26±0.83cm, 2.94±0.75cm, and- 0.80±0.25cm, respectively) and during contraction (3.95±0.78cm, 2.71±0.78cm, and 0.87±0.29cm, respectively) in the affected side than in the healthy side (5.45±0.66cm, 3.87±0.49cm, and 0.97±0.20cm, respectively at rest; 4.99±0.45cm, 3.49±0.53cm, and 1.07±0.23cm, respectively during contraction, p < 0.05). In the resting state, the hardness of the masseter muscle on the affected side (0.77±0.66m/s) was significantly greater than that on the healthy side (0.42±0.41m/s) (p <0.05). The magnitude of changes in the upper-lower, left-right, and anterior-posterior diameters of the biting muscle in the occlusal state were significantly smaller on the affected side (-0.30±0.27cm, -0.23±0.17cm, and 0.08±0.08cm, respectively) than in the healthy side (-0.47±0.38cm, -0.37±0.25cm, and 0.10±0.12cm, respectively, p<0.05) ConclusionsThe knowledge of these values allows for a better understanding of the disease characteristics of HFM, which may be used for its diagnosis, treatment, and prognosis.Patients of different severity levels exhibited significant differences in both the morphology and function of the affected-side masseter muscle (p < 0.05). Evidence levelLevel III