Objective: Chronic stress and repeated physiological attempts at stress adaptation may result in 'fatigue' and suboptimal performance of multiple physiological systems, i.e. allostatic load (AL). Although carotenoids have been linked with individual cardiovascular, metabolic and inflammatory biomarkers, little is known about the relationship of carotenoids with the multi-system biomarker measure of stress, AL. The present study examined the association of serum concentrations of carotenoids with AL among middle-aged adults. Design: Cross-sectional. AL score was calculated based on nine risk-rated indicators (systolic and diastolic blood pressure, pulse rate, total and HDL-cholesterol, glycosylated Hb, sex-specific waist-to-hip ratio, albumin and C-reactive protein). Subjects: Middle-aged (45-64 years, n 3387) men and women participants in the Third National Health and Nutrition Examination Survey, NHANES III (1988-1994. Results: Serum b-carotene concentration was inversely associated with high AL after adjusting for age, education, race/ethnicity, serum cotinine, alcohol consumption, physical activity and other carotenoids (a-carotene, b-cryptoxanthin, lycopene, lutein/zeaxanthin). Females in the lowest b-carotene quartile were 2?94 (95 % CI 1?74, 4?94) times and males 2?90 (95 % CI 1?43, 5?89) times as likely to have high AL, compared with peers in the highest quartile (P for linear trend 0?001 and 0?018 for females and males, respectively). Mean serum b-carotene concentrations were also inversely associated with the number of 'high-risk' AL components (P for linear trend ,0?001 and 0?004 for females and males, respectively). Conclusions: Our study adds to evidence linking low b-carotene levels with unfavourable health outcomes.