Introduction Time-of-day has been suggested as a contributing factor to sensitivity to pain. However, inconsistent findings have been found regarding the specific time-of-day in which sensitivity to pain is at its peak. Methodological differences in study settings and samples preclude firm conclusions to be drawn, leaving this issue unresolved. The present study aimed to assess the effect of time-of-day on sensitivity to experimentally evoked pain in healthy subjects exposed to multi modal pain tests. It was hypothesized that time-of-day response patterns to pain would demonstrate stability across pain modalities. Materials and methods Forty-eight healthy men were tested in the morning, early afternoon and evening in a randomized order repeated measures design. Four pain threshold measures: mechanical (pressure algometer), heat (thermal sensory analyzer) and cold (thermal sensory analyzer and cold bath) were assessed in each session, and were compared by repeated measures ANOVA with Bonferroni post hoc tests to assess specific time-of-day differences. Also, pain threshold measures were standardized and hierarchical clustering and K-means cluster analyses were performed to identify subgroups of low vs. high sensitivity subjects by time-of- day. MANOVA was performed to validate differences in pain thresholds by clusters. Results Significant differences were found for the two cold pain thresholds, with lowest sensitivity in the morning; and for the heat pain threshold, with highest sensitivity in the afternoon (p<0.05). No differences were found for the mechanical pain threshold. Cluster analyses varied by time-of-day, so that the high sensitivity subgroup increased from 50% of the subjects in the morning to 75% in the afternoon and 86% in the evening. For all times of day, significant differences in all pain thresholds were found between low and high sensitivity clusters (p<0.05). Conclusion Sensitivity in different pain modalities displays different time-of-day patterns, yet the overall frequencies of pain sensitivity increase throughout the day, with lowest frequency in the morning and highest frequency in the evening. Whether time-of-day effects in pain sensitivity reflect underlying circadian and/or homeostatic sleep/wake mechanisms is yet to be determined. Sub-grouping subjects based on sensitivity to pain should take into account time-of-day differences. Findings may provide valuable information for clinical trials on chronic pain patients. Acknowledgement The study was funded by a grant from the Israel Pain Society.