Repeated heat pain threshold testing in pain-free individuals induces habituation and yields false-positive pre-post comparisons.

Heat pain thresholds (HPTs) are used for clinical assessments, sensory phenotyping, and pre-post comparisons. This usage assumes that HPTs are stable, exchangeable, and unaffected by repetition. Twenty consecutive HPT measures were taken at each volar forearm during three daily sessions in pain-free individuals. Negative exponential growth models were used to characterise HPT series and estimate effects of session, side, and time between sessions. To assess suitability for within-session pre-post comparisons, the mean of the first n repetitions of a series was compared with the mean of the subsequent n repetitions for n = 1-10, and ICC(3,1) calculated for n = 2-20. From 23 participants, 2266 HPT measurements were recorded within 118 series. Strong habituation was observed (Cohen's d = 1.9), with HPT increasing by a mean of 4.9 °C across all series (95% CI: 4.4 to 5.3 °C, p <0.001). Repeated HPTs followed an asymptotic trajectory; lower initial HPTs were associated with larger repetition-related increases, and right forearms adapted faster than left. Later sessions were associated with higher initial and asymptotic HPT values and faster adaptation. Using single or averaged HPTs for within-series pre-post comparisons consistently yielded false-positive findings, while ICC decreased as n increased.