Aims of investigation: To quantify the magnitude of putative gender differences in experimental pressure pain threshold (PPT), and to establish the relevance of repeated measurements to any such differences. Methods: Two separate studies were undertaken. A pressure algometer was used in both studies to assess PPT in the first dorsal interosseous muscle. Force was increased at a rate of 5 N /s. In study 1, two measurements were taken from 240 healthy volunteers (120 males, 120 females; mean age 25 years) giving a power for statistical analysis of β=0.80 at α=0.01. In study two, 30 subjects (15 males, 15 females mean age 28 years) were randomly selected from study one. Fourteen repeated PPT measurements were recorded at seven, 10 min intervals. Mean PPT data for gender groups, from both studies, were analysed using analysis of covariance with repeated measures, and age as the covariate. Results: The mean PPT for each of the two measurements in study one showed a difference between gender of 12.2 N (f=30.5 N, m=42.7 N) and 12.8 N (f=29.5 N, m=42.3 N), respectively, representing a difference of 28% with females exhibiting a lower threshold. In study two, the mean difference calculated from 14 PPT repeated measurements over a 1 h period was comparable to that in study one at 12.3 N (range 10.4–14.4 N) again females exhibited the lower threshold. The differences in mean PPT values between gender were found to be significant in both study one, at (P<0.0005, F=37.8, df=1) and study two (P=0.01, F=7.6, df=1). No significant differences were found in either study with repeated measurement (P=0.892 and P=0.280), or on the interaction of gender and repeated measurement after controlling for age (P=0.36 and P=0.62). Conclusion: Healthy females exhibited significantly lower mean PPTs in the first dorsal interosseous muscle than males, which was maintained for fourteen repeated measures within a 1 h period. This difference is likely to be above clinically relevant levels of change, and it has clear implications for the use of different gender subjects in laboratory based experimental designs utilising PPT as an outcome measure.