Background: Repeated injection of acidic saline into skeletal muscles of the leg in rodents induces a prolonged bilateral mechanical hyperalgesia that persists for up to 30 days and may be useful to model widespread muscle pain conditions. In this study, repeated injection of acidic (pH 3.3) saline solution into the masseter muscle of healthy human subjects was undertaken to determine if these injections are painful and whether they would induce a prolonged period of muscle sensitization to artificial and/or natural mechanical stimulation of the masseter and temporalis muscles. Methods: Eighteen subjects (10 male, 8 female) participated in the study. Subjects received two injections of 0.5 mL acidic or regular isotonic saline 2 days apart, in a randomized, double blind, crossover design. Results: There was no significant difference in pain intensity ratings when acidic saline injections were compared with regular saline injections. Pain area drawings were, however, significantly larger in response to the first injection of acidic saline than to the second injection of acidic saline or to either the first or second injection of regular saline. Repeated injection of acidic saline did not significantly alter pressure pain thresholds from the masseter or temporalis muscles on either the injected side or the opposite side over the 10-day post injection monitoring period. There was also no effect of injections on chewing. Conclusion: These findings indicate that, unlike in some rodent models, repeated injection of low pH solutions into jaw muscles of humans fails to induce a period of prolonged muscle hyperalgesia. © 2013 European Federation of International Association for the Study of Pain Chapters.