The hot water tail-flick test is widely used to measure the degree of nociception experienced by laboratory animals. This study was carried out to optimise interval times for the hot water immersion tail-flick tests in rats.

Ten different intervals from 10 s to 1 h were tested in 60 Sprague–Dawley male rats. At least eight rats were tested for each interval in three consecutive hot water tail-flick tests. Dixon's up-and-down method was also used to find the optimal intervals. The same rats were then divided into two groups. In Group N, naloxone was injected to reverse the prolonged latency times, whereas saline was used in the control Group S.

Intervals of 10 s, 20 s, 30 min and 1 h did not significantly impact latencies, yielding similar results in three consecutive tests (p > 0.05). However, interval times of between 30 s and 20 min, inclusively, caused significantly prolonged latencies in the second and third tests (p < 0.001). Dixon's up-and-down method showed that 95% of the rats had prolonged latencies in hot water tail-flick tests at intervals longer than 32 s. Naloxone reversed prolonged latencies in Group N, whereas the latencies in Group S were further prolonged in 5 min interval tests.

The optimal intervals for hot water tail-flick tests are either shorter than 20 s or longer than 20 min. The prolonged latencies after repetitive tests were attributable to an endocrine opioid.