Aims: To investigate whether experimental tooth clenching leads to a release of algesic substances in the masseter muscle. Methods: Thirty healthy subjects (16 females, 14 males) participated. During two sessions, separated by at least 1 week, intramuscular microdialysis was performed to collect masseter muscle 5-hydroxytryptamine (5-HT) and glutamate as well as the metabolic markers pyruvate and lactate. Two hours after the start of microdialysis, participants were randomized to a 20-min repetitive experimental tooth-clenching task (50% of maximal voluntary contraction) or a control session (no clenching). Pain and fatigue were measured throughout. The Friedman and Wilcoxon tests were used for statistical analyses. Results: No alterations were observed in the concentrations of 5-HT, glutamate, pyruvate, and lactate over time in the clenching or control session, or between sessions at various time points. Pain (P .01) and fatigue (P .01) increased significantly over time in the clenching session and were significantly higher after clenching than in the control session (P .01). Conclusion: Low levels of pain and fatigue developed with this experimental tooth-clenching model, but they were not associated with an altered release of 5-HT, glutamate, lactate, or pyruvate. More research is required to elucidate the peripheral release of algesic substances in response to tooth clenching.