Objective: The observed poorer physical function in persons with mental disorders could partly be due to dysregulation in physiological stress systems. However, an integrated picture of the role of physiological stress systems on objective physical function is lacking. This study examined the association of multiple physiological stress systems with objective physical function, and explored whether these stress systems contribute to the relationship between depression/anxiety and poorer physical function. Methods: Data of 2860 persons of the Netherlands Study of Depression and Anxiety was used. Physical function was indicated by hand grip strength assessed using a hand-held dynamometer and lung function assessed using a peak flow meter. Inflammatory markers (CRP, IL-6, TNF-α), salivary cortisol (cortisol awakening response (AUCg, AUCi), evening cortisol) and ANS markers (heartrate, PEP, RSA) were determined. Depression/anxiety disorders were determined using psychiatric interviews. Linear regression analyses were adjusted for sociodemographics, health and lifestyle factors. Results: Higher inflammation levels were associated with lower hand grip strength (B CRP = -0.21(SE = 0.06), p < .001) and lower lung function (B CRP = -2.07(SE = 0.66), p = .002), B TNF-α = −3.35(SE = 1.42), p = .022). Higher salivary cortisol levels were associated with lower lung function (B evening cortisol = −2.22(SE = 0.59), p < .001). The association, in women, between depression/anxiety disorders and poorer physical function did not significantly diminish after adjustment for physiological stress markers. Conclusion: This large cohort study showed that stress system dysfunction (especially the immune-inflammatory system and HPA-axis) contributes to poorer objective physical function. Stress system dysfunction did not explain the poorer physical function observed in persons with depression/anxiety disorders, suggesting that other pathways are involved to explain that association.