Background: Animal studies have shown that two repeated intramuscular injections of acidic saline induce mechanical allodynia that lasts for 4 weeks with spread to the contralateral side. In this study, we tested the hypothesis that two repeated intramuscular infusions of acidic saline into the human masseter muscle is associated with pain, mechanical allodynia and release of algesic substances. Eighteen healthy volunteers participated. On day 1, 2.5 mL of acidic saline (pH 3.3) was infused into one of the masseter muscles and isotonic saline (pH 6.0) into the other (randomized and single-blind). Two days later, intramuscular microdialysis was performed to sample serotonin, glutamate, pyruvate, lactate and glucose, during which the saline infusions were repeated. Pain and pressure pain thresholds (PPTs) were recorded before and after infusions on both days. Results: Pain intensity induced by the infusions was higher after acidic than that after isotonic saline (p < 0.05). PPTs were decreased on both sides after microdialysis compared with baseline day 1 (p's < 0.05), but there were no differences in PPTs between sides at any time point. The levels of serotonin, glutamate, pyruvate, lactate or glucose did not change significantly during microdialysis. Conclusion: Infusion of acidic saline caused low levels of muscle pain, but no mechanical allodynia and no increased release of algesic substances. The value of this model appears modest, but future studies could be performed with larger sample size and higher flow rate before definite conclusions about the validity of the model for craniofacial myalgia can be drawn. © 2012 European Federation of International Association.