Background Conditioned pain modulation (CPM) and offset analgesia quantify impairment of endogenous pain modulation, but magnitude and reliability vary broadly between studies, indicating influencing factors that are not currently controlled for. The aim of this study was to quantify magnitude and retest reliability of CPM and offset analgesia in healthy participants, while investigating the influence of sex and sex hormone levels. Methods Sixty‐two participants (30 female) completed the study. We tested CPM (heat–cold paradigm) and offset analgesia on 6 days within two menstrual cycles (tests were performed in each phase of two subsequent menstrual cycles, with similar time points for men). Results Median offset effect was −29.4% in female and − 22.5% in male participants (as change from initial stimulus). Median early CPM effects were − 16.7% for women versus −13.3% for men. Reliability (intra‐class correlation coefficient [ICC]) was similar between the main measures, offset effect (female: 0.48, male: 0.47) and early CPM effect (female: 0.49, male: 0.43). There was significant variance between individual experimental parameters within protocols but not between sexes or menstrual phases. While oestradiol and progesterone did not correlate with the magnitude of effect within sexes, we found that testosterone levels explained an estimated 5%–10% of variance within individual responses in all sexes. Conclusions Our results show that the reliability of both CPM effect and offset analgesia was independent of sex and menstrual cycle phase. The magnitude of CPM and offset effects was weakly influenced by sex and testosterone levels, indicating an area for future research, rather than clinical significance. Significance This study investigated CPM and offset analgesia in parallel, across sexes and during two menstrual cycles while assessing the impact of sex hormones. Reliability seems to depend on experimental parameters rather than participant characteristics, while the magnitude of effect could be weakly linked to sex hormones and sex.