Context: The ’normal‘ cortisol response to an ACTH-test remains inconsistently defined, possibly caused by various subject- and test condition-related factors.

Objective: To evaluate the impact of: newer automated immunoassays; gender, age, body composition, and endogenous sex-hormone levels; corticosteroid binding globulin (CBG) levels; and test-conditions (fasting/non-fasting, rest/intermittent exercise).

Methods: A 250µg ACTH-test (0800-1000) was performed in 100 unmedicated subjects, 13 women taking oral contraception (OC) and 6 men with nephrotic syndrome (NS). Tests were performed fasting supine(n=119), non-fasting supine(n=38) and fasting with intermittent exercise(n=45). Se-cortisol was analyzed by three immunoassays.

Results: Even with a negligible between-assay mean bias, individual samples from unmedicated subjects differed as much as 110 nmol/L. The normative 2.5^th percentile for total-cortisol ranged from 475-523 nmol/L, when analyzed by the three assays. In multivariate analyzes, 30 min total-cortisol was predicted by baseline cortisol (men+women) and central adiposity (men), but not by gender, age, and endogenous sex-hormones; CBG; fasting/non-fasting; and exercise. Compared to unmedicated subjects, OC-women had 2-fold elevated 30 min cortisol (p<0.001) but lowered CFC (p<0.001), whereas NS-patients had lowered 30 min cortisol (p<0.01) in two of three assays, but similar CFC (p>0.1).

Conclusion: The ’normal‘ response to an ACTH-test is assay specific, even with newer methods and this also applies to calculated free cortisol. Both total cortisol and calculated free cortisol were severely affected by OC, and the test is therefore only reliable if OC has been discontinued. The ACTH-test is however robust as concern most of the other evaluated subject- and test condition- related factors.