Aims: To evaluate the contraction pattern of masticatory muscles during sustained clenching tasks with or without experimental pain induced by glutamate injection into the masseter muscle. It was hypothesized that acute muscle pain could induce compensatory changes in the electromyographic (EMG) activity of the masticatory muscles. Methods: Fifteen volunteers (seven males, mean age ± SD = 29.7 ± 1.1 years; eight females, mean age ± SD = 23.5 ± 1.2 years) were recruited in a crossover experimental study. All subjects participated in two randomized 20-minute experimental sessions. Each subject was asked to clench at 25% of the maximum voluntary contraction (MVC). After 10 minutes, isotonic saline or glutamate was injected in random order into the right masseter. EMG activity (root mean square [RMS] and mean power frequency [MPF]) was assessed in the masseter and anterior temporalis muscles on both sides. Pain and fatigue were assessed by 0-10 numeric rating scales (NRS) every minute. Differences between conditions (isotonic saline vs glutamate) for all the outcome parameters were analyzed by using a mixed effect model. Results: The EMG activity of the masticatory muscles and pain and fatigue scores were not dependent on isotonic saline/glutamate injection (all P > .05). The RMS in the temporalis and masseter muscles increased with time (right masseter P = 0.001, left masseter P = .004, right temporalis P = .22, left temporalis P = .006), whereas the MPF decreased (right masseter P = 0.0001, left masseter P .0001, right temporalis P = 0.51, left temporalis P = .0005). Scores for fatigue and pain increased during the experimental sessions (all P .05). Conclusion: Intramuscular injection of glutamate caused more pain than isotonic saline but did not affect the contraction pattern of the masticatory muscles during a sustained clenching task. This finding strongly suggests the adaptive capacity of the stomatognathic system in the presence of acute nociceptive inputs. Keywords: experimental orofacial pain, surface electromyography, temporomandibular disorders, tooth clenching