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The aim of this study was to determine whether salivary cortisol measured by a simple enzyme immunoassay (EIA) could be used as a surrogate for serum total cortisol in response to rapid changes and across a wide range of concentrations.Comparisons of matched salivary and serum samples in response to dynamic hypothalamic–pituitary–adrenal (HPA) axis testing.Healthy women (n = 10; three taking oral oestrogens) and men (n = 2), aged 23–65 years, were recruited from the community.Paired saliva and serum samples were obtained during three protocols: 10 min of exercise at 90% of maximal heart rate (n = 8), intravenous administration of corticotrophin-releasing hormone (CRH; n = 4), and dexamethasone suppression (n = 7). Cortisol was measured in saliva using a commercial high-sensitivity EIA and total cortisol was measured in serum with a commercial radioimmunoassay (RIA).The time course of the salivary cortisol response to both the exercise and CRH tests paralleled that of total serum cortisol. Salivary cortisol demonstrated a significantly greater relative increase in response to the exercise and CRH stimuli (697 ± 826%vs. 209 ± 150%, P = 0·04 saliva vs. serum). A disproportionately larger increase in free cortisol, compared with total, would be expected when the binding capacity of cortisol-binding globulin (CBG) is exceeded. In response to dexamethasone suppression, relative decreases in cortisol were not significantly different between the two media (−47 ± 56%vs.−84 ± 8%, P = 0·13 saliva vs. serum). Although a significant linear correlation was found for all paired salivary and serum total cortisol samples (n = 183 pairs, r = 0·60, P < 0·001), an exponential model provided a better fit (r = 0·81, P < 0·001). The linear correlations were strengthened when data from subjects on oral oestrogens (n = 52 pairs, r = 0·75, P < 0·001) were separated from those not taking oestrogens (n = 131 pairs, r = 0·67, P < 0·001).Salivary cortisol measured with a simple EIA can be used in place of serum total cortisol in physiological research protocols. Evidence that salivary measures represent the biologically active, free fraction of cortisol includes: (1) the greater relative increase in salivary cortisol in response to tests that raise the absolute cortisol concentration above the saturation point of CBG; (2) the strong exponential relationship between cortisol assessed in the two media; and (3) the improved linear correlations when subjects known to have increased CBG were analysed separately. Thus, an advantage of measuring salivary cortisol rather than total serum cortisol is that it eliminates the need to account for within-subject changes or between-subject differences in CBG.