Several human models of myofascial pain exist, but none are similar to clinical pain. The aim of the present study was to develop a clinically relevant model of prolonged human myofascial pain using infusion of the naturally occurring endogenous substances. Initially, bradykinin (Bk), serotonin (5-hydroxytryptamine (5-HT)), histamine (His), prostaglandin E₂ (PGE₂), adenosine-tri-phosphate (ATP), and their combinations were infused into the trapezius muscle of 36 healthy subjects in a total of 67 sessions to identify substances, which could induce a moderate muscle pain. PGE₂, ATP, and a combination of Bk, 5-HT, His, and PGE₂ produced the intended moderate pain. These substances were further examined in a randomised, blinded, placebo-controlled dose-finding design in 15 healthy subjects in 68 sessions. PGE₂ (3, 6, and 12nmol/ml) induced mild pain and tenderness not different from placebo. ATP (9000, 18,000, and 36,000nmol/ml) induced pain of moderate to strong intensity (P=0.04) and the dose of 18,000nmol/ml furthermore produced moderate local tenderness (P=0.04). Because of unacceptable side effects in subsequent examinations, further studies of ATP in humans were suspended. Infusion of the combination of Bk (92nmol), 5-HT (156nmol), His (140nmol), and PGE₂ (1.95nmol) produced a moderate pain intensity (P=0.04) and mild tenderness (P=0.04) without inducing unacceptable side effects. Intramuscular infusion of a combination of Bk, 5-HT, His, and PGE₂ induced a prolonged moderate pain and tenderness in healthy humans, and this model may be a valuable tool in future studies of the pathophysiological mechanisms of myofascial pain.