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Intramuscular injection of hypertonic saline is a good model to study human muscle pain (Kellgren 1938). The present study concerns the intramuscular (i.m.) pain mediators in saline-induced muscle pain. In experiment 1, the diffusion of infused hypertonic and isotonic saline (0.5 ml) in m. tibialis anterior was illustrated by magnetic resonance imaging (MRI) in one subject. In experiment 2, six volunteers received four sequential infusions (0.5 ml given at 5 min intervals) of isotonic saline and thereafter four sequential infusions (0.5 ml given at 5 min intervals) of hypertonic saline into m. tibialis anterior. The isotonic and hypertonic saline infusions were computer-controlled and separated by 20 min. The muscle pain intensity was assessed by continuous recordings on a visual analogue scale (VAS). One microdialysis probe was inserted 1 cm from the infusion needle in m. tibialis anterior and another probe in the other m. tibialis anterior. Concentrations of the i.m. sodium, potassium, magnesium, and prostaglandin E2 (PGE2) were assessed from the dialysates. Intramuscular electromyography (EMG) and pressure were assessed in the area of the infused saline. In experiment 1, the infusion of hypertonic and isotonic saline created a visible saline-pool on the MRI scans. These saline-pool volumes were stable and not correlated to the pain scores. In experiment 2, infusion of isotonic saline produced little pain compared to infusion of hypertonic saline. Maximal pain was reported after the first infusion of hypertonic saline and thereafter the pain gradually decreased with subsequent infusions of hypertonic saline. During infusion of hypertonic saline the i.m. sodium and potassium concentrations increased significantly, i.m. magnesium concentration tended to be increased, and the i.m. PGE2 concentration tended to be decreased although these changes were not significant. The i.m. EMG was smaller during and after infusions of hypertonic saline compared with isotonic saline. The i.m. pressure was not different during the infusions of hypertonic and isotonic saline but was increased between the infusions of hypertonic saline. This study has shown that i.m. infusion of hypertonic saline produced a saline-pool, causing the i.m. pressure to increase. Possibly, pain activation and cessation are related to increased intramuscular sodium and potassium content respectively.