Reclassification of the human masseter muscle: anatomical, histological, and ultrasonographic characterization of its deep layer

BACKGROUND: Recent studies have challenged the traditional two-layer description of the masseter, suggesting a distinct innermost layer. Clarifying the identity of this structure requires integration of macroscopic, histological, and functional perspectives. MATERIALS AND METHODS: We examined 22 Korean cadavers using lateral and medial dissection routes. We prepared transverse histological sections (H&E and Masson's trichrome) and performed ultrasonography at standardized anatomical landmarks. Morphometric variables were recorded, and associations with sex and age were analyzed. RESULTS: The innermost layer originated from the medial surface of the zygomatic arch and inserted onto the mandibular notch and posterior coronoid process. Well-defined fascial planes separated this layer from the temporalis and from the traditional deep portion of the masseter, designated here as the middle part. Comparative anatomy linked this layer to the maxillomandibularis in nonhuman primates. Quantitative analyses showed pronounced sexual dimorphism and progressive age-related atrophy. CONCLUSIONS: We propose redefining the masseter as a three-layer muscle - superficial, middle (formerly deep), and deep (the newly characterized layer). This framework increases anatomical precision and evolutionary continuity and may provide a more reliable basis for future functional studies and clinical applications.