To investigate the effects on pain, movement kinematics, and cerebral representation by a 3-month mandibular splint therapy.Thirteen patients with temporo-mandibular joint disease (TMD) and moderate pain intensity were investigated before (PRE), within (after 2 weeks, POST1) and after a period of 12 weeks (POST2) using functional magnetic resonance imaging (fMRI) of representation of occlusal movements on natural teeth and on an individually fitted mandibular splint. In addition, kinematic investigations of jaw movements, muscle electromyography and pain ratings using a pain diary (VAS-scale 0-100) were measured.Although the patient's pain ratings decreased about 60%, kinematic and electromyographic characteristics over therapy were not significantly altered. Over therapy, we observed a decrease of fMRI activation magnitude in the primary somatosensory cortex (S1) and secondary somatosensory cortex (S2) and insular cortex during occlusion. Left hemispheric anterior insula and the cerebellar fMRI activation decrease were associated with decrease in pain over time.Within the limitations of this pilot study, a reduction in both discriminative (primary and secondary somatosensory cortex) and affective (anterior insula) areas for pain processing suggest that altered pain anticipation is critical for the therapeutic effects of mandibular splint therapy after TMD.A 3-month mandibular splint therapy moderately decreases pain and anticipatory anterior insular activation.