Most of the research concerning magnetic antinociception was focused on brief exposure less than 1 h. The main purpose of the present study was to determine the effect of extremely low frequency (ELF) magnetic field (MF) repeated exposures on rats in inducing antinociception and to find the effective analgesic "time window." Meanwhile this investigation was to examine the role of central beta-endorphin, substance P, and 5-HT in magnetic analgesia. We found tail flick latencies (TFLs) increased significantly after the rats were exposed to 55.6 Hz, 8.1 mT magnetic field for 4 days, 6 h each day. The analgesic effects seemed to decrease gradually when the rats were exposed daily for another 10 days. Their levels of TFLs decreased within 1 day when the rats were removed after a 4-day exposure. The concentrations of hypothalamus beta-endorphin, substance P, and brainstem serotonin (5-HT) were increased significantly on Day 4. However, no differences were found when rats were exposed for another 10 days, and there were no significant increases when rats were removed after the fourth day of exposure and tested for nociception on Days 5 and 7 with no changes in the biochemical markers at 7 days. These results suggest that the ELF magnetic field has analgesic effect, but only on Days 3 and 4. The effect may be associated with increases in endogenous beta-endorphin, substance P, and 5-HT stimulated by the 55.6 Hz, 8.1 mT magnetic field.