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Allostatic load (AL) reflects the deteriorating influences of stress on the body and comprises a selection of biological markers. AL is associated with negative life events, stress, and negative affect (NA), as well as poor health outcomes. However, whether AL is also associated with positive affect (PA) is not clear. The present study therefore explores the association between PA and AL, accounting for age, sex, NA, and health behaviors.Data of 45,225 individuals from the first wave of the multidisciplinary prospective population-based cohort study Lifelines were used. AL was operationalized as the sum of 12 inflammatory, cardiovascular, and metabolic markers. The association between PA and AL was tested in a cross-sectional study design using multiple linear regression analysis, adjusting for NA, confounders, and health behaviors. In addition, we explored whether the relation was moderated by age, sex, and NA.The AL profile was inversely associated with PA (B = −0.083, p < .001) when adjusted for NA, age, and sex. The association between AL and PA remained significant after adjusting for health behaviors (B = −0.076, p < .001). A significant moderating effect was found for sex (PA by sex: B = 0.046, p = .001), indicating that the association between PA and AL was stronger in women than in men.PA was associated with a more favorable AL profile, especially in women. These results add to the evidence that PA might be of relevance to the etiology of disease.