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Background and aims There is high level evidence for physical activity (PA) improving outcomes in persistent pain disorders and one of the mechanisms proposed is the effect of exercise on central nociceptive modulation. Although laboratory studies and small field intervention studies suggest associations between physical activity and pain sensitivity, the association of objectively measured, habitual PA and sedentary behaviour (SB) with pain sensitivity requires further investigation. Current evidence suggests PA typically lowers pain sensitivity in people without pain or with single-site pain, whereas PA is frequently associated with an increase in pain sensitivity for those with multisite pain. The aim of this study was to explore the relationships of PA and SB with pain sensitivity measured by pressure pain thresholds and cold pain thresholds, considering the presence of single-site and multisite pain and controlling for potential confounders. Methods Participants from the Western Australian Pregnancy Cohort (Raine) Study (n = 714) provided data at age 22-years. PA and SB were measured via accelerometry over a 7-day period. Pain sensitivity was measured using pressure pain threshold (4 sites) and cold pain threshold (wrist). Participants were grouped by number of pain areas into "No pain areas" (n = 438), "Single-site pain" (n = 113) and "Multisite pain" (n = 163) groups. The association of PA and SB variables with pain sensitivity was tested separately within each pain group by multivariable regression, adjusting for potential confounders. Results For those with "Single-site pain", higher levels (>13 min/day) of moderate-vigorous PA in ≥10 min bouts was associated with more pressure pain sensitivity (p = 0.035). Those with "Multisite pain" displayed increased cold pain sensitivity with greater amounts of vigorous PA (p = 0.011). Those with "No pain areas" displayed increased cold pain sensitivity with decreasing breaks from sedentary time (p = 0.046). Conclusions This study was a comprehensive investigation of a community-based sample of young adults with "No pain areas", "Single-site pain" and "Multisite pain" and suggests some associations of measures of PA and SB with pain sensitivity. Implications The findings suggest that the pattern of accumulation of PA and SB may be important to inform improved clinical management of musculoskeletal pain disorders. This study provides a baseline for follow-up studies using the Raine Study cohort. Future research should consider temporal influences of PA and SB on pain sensitivity, pain experience and consider using a broader range of pain sensitivity measures.