Objective: The objective of the present study is to investigate if changes in the oxygen saturation of masseter muscle during a chewing task can differentiate patients with myogenic temporomandibular disorders (TMD) from healthy subjects and if these differences are related to the gravity of the disorder and to the orofacial myofunctional status. Materials and methods: Twelve women with moderate TMD (TMD group; 37 ± 16 years) and ten healthy control women (CTRL group 24 ± 5 years) participated. Validated protocols were used to evaluate the severity of TMD and the orofacial myofunctional status. Oxygen saturation in the masseter muscle was measured using near-infrared spectroscopy (NIRS) during unilateral chewing of a silicon device. Data were compared using Student’s t test, Mann-Whitney test, and Spearman’s rank correlation coefficient. Results: The women of the TMD group showed higher total score of severity of symptoms of TMD, lower total score of the orofacial myofunctional status, and lower oxygen extraction capacity during mastication than healthy control subjects (p < 0.01). Moreover, percentage O2 extraction was significantly related to the severity of signs/symptoms of TMD and of orofacial myofunctional disorders (p < 0.01). Conclusion: Women with TMD had a lower muscle oxygen extraction capacity than healthy subjects: the higher the signs and symptoms' severity, the lower the O2 extraction. NIRS proposes as an important instrumental method to assess the metabolic alterations in the muscles of patients with TMD. Clinical relevance: The findings could be useful to complement clinical assessments, favoring the diagnosis and providing extra data for planning the rehabilitation of TMD patients, especially those with associated myofunctional orofacial disorders.