Objective: The purpose of this study was to assess the effect of upper thoracic manipulation on vertical mouth opening (VMO) and electromyographic activity of the masticatory muscles in women with temporomandibular disorder (TMD). Methods: A randomized, controlled, blinded study involving 32 women (mean age of 24.78 ± 5.41 years) diagnosed with TMD was performed. Subjects were randomly allocated to an experimental group (n = 16) or a sham manipulation (placebo) group (n = 16). A single session of manipulation was administered to the T1 vertebral area. Electromyography was used to determine activity of masticatory muscles with the mandible at rest and during isometric contraction. Vertical mouth opening was measured using a ruler. Measurements were made before manipulation, immediately after manipulation, and at the short-term follow-up (2 to 4 days after manipulation). Results: No significant differences were found in any of the comparisons of mandibular VMO or electromyographic activity of the masticatory muscles at rest or during isometric contraction of mandibular elevator muscles (P > .05). Regarding isometric contraction of the mandibular depressors, an immediate increase in muscle activity was found in the suprahyoid (P = .014) and left masseter (P = .005) muscles. Conclusion: In the present study, no significant differences were found regarding VMO between the experimental and placebo groups or among the different evaluations times in each group. Moreover, Cohen d test revealed no clinical effect of the technique. Therefore, based upon the findings of this study, manipulation administered to the T1 vertebral area appears to have no effect on VMO and electromyographic activity of the masticatory muscles in women with TMD.