The aim of this study was to investigate the surface electromyographic (sEMG) activity of neck, trunk, and masticatory muscles in subjects with temporomandibular joint (TMJ) internal derangement treated with anterior mandibular repositioning splints. sEMG activities of the muscles in 34 adult subjects (22 females and 12 males; mean age 30.4 years) with TMJ internal derangement were compared with a control group of 34 untreated adults (20 females and 14 males; mean age 31.8 years). sEMG activities of seven muscles (anterior and posterior temporalis, masseter, posterior cervicals, sternocleidomastoid, and upper and lower trapezius) were studied bilaterally, with the mandible in the rest position and during maximal voluntary clenching (MVC), at the beginning of therapy (T0) and after 10 weeks of treatment (T1). Paired and Student's t-tests were undertaken to determine differences between the T0 and T1 data and in sEMG activity between the study and control groups. At T0, paired masseter, sternocleidomastoid, and cervical muscles, in addition to the left anterior temporal and right lower trapezius, showed significantly greater sEMG activity (P = 0.0001; P = 0.0001; for left cervical, P = 0.03; for right cervical, P = 0.0001; P = 0.006 and P = 0.007 muscles, respectively) compared with the control group. This decreased over the remaining study period, such that after treatment, sEMG activity revealed no statistically significant difference when compared with the control group. During MVC at T0, paired masseter and anterior and posterior temporalis muscles showed significantly lower sEMG activity (P = 0.03; P = 0.005 and P = 0.04, respectively) compared with the control group. In contrast, at T1 sEMG activity significantly increased (P = 0.02; P = 0.004 and P = 0.04, respectively), but no difference was observed in relation to the control group. Splint therapy in subjects with internal disk derangement seems to affect sEMG activity of the masticatory, neck, and trunk muscles.