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Background: Botulinum toxin (BoNT) is widely used to treat masseter muscle hypertrophy. Changes in the muscle thickness have been found in many studies, but there has been no report on changes in the thickness from the skin surface to the masseter muscle.Objectives: We aimed to use ultrasonography to measure not only changes in the muscle thickness but also changes in subcutaneous thickness.Methods: This study enrolled 20 volunteer patients: 10 were assigned to an experimental group (injected with each side 25 U of botulinum toxin into both masseter muscles) and 10 to a control group (injected with normal saline). The thicknesses were measured before the injection and at 4, 8, and 12 weeks after the injection both at rest and during maximum muscle contraction.Results: The subcutaneous thickness did not differ significantly over time either at rest (P = 0.063) or during maximal contraction (P = 0.392), or between the experimental and control groups at rest (P = 0.392) or during maximum contraction (P = 0.259). The muscle thickness in the experimental group differed significantly over time.Conclusions: Botulinum toxin injection only changes the muscle thickness and does not affect the subcutaneous thickness from the skin surface to the masseter muscle. [ABSTRACT FROM AUTHOR]