BACKGROUND: Cross-sectional studies have suggested that elevated cortisol is associated with worse physical performance, a surrogate of ageing. We examined the relationship between repeat cortisol measures over 20 years and physical performance in later life. METHODS: Middle-aged men (45-59 years) were recruited between 1979 and 1983 (Phase 1) from the Caerphilly Prospective Study (CaPS) and re-examined 20 years later at 65-83 years of age (Phase 5). Participants included 750 and 898 subjects with either Phase 1 and/or Phase 5 data on exposure and outcomes. Outcome measures were walking speed and balance time and exposures included morning fasting serum cortisol (Phase 1) and four salivary samples on 2 consecutive days (Phase 5). RESULTS: Faster walking speed was associated with higher morning cortisol at Phase 1 [coefficient per standard deviation (SD) increase 0.68, 95% confidence interval (95% CI) 0.09-1.27; P=0.02] though this was attenuated after adjustment for covariates (coefficient per SD increase 0.45; 95% CI -0.16 to 1.07; P=0.15). Higher night-time cortisol at Phase 5 was associated with slower speed (coefficient per SD increase -1.06; 95% CI -1.60 to -0.52; P<0.001) and poorer balance (odds ratio of top tertile vs bottom 2.49; 95% CI 1.63-3.81; P<0.001). Worst performance was seen for men with a poor morning response (Phase 1) and less nocturnal decline (Phase 5). CONCLUSIONS: Dysregulation of the hypothalamic pituitary adrenal (HPA) axis is associated with worse physical performance in later life. This may reflect a causal effect of the HPA axis on ageing or that ageing itself is associated with reduced HPA reactivity.