Background and aim <p>Monoclonal immunoassays offer greater specificity than polyclonal assays for cortisol measurement, necessitating assay-specific cut-off limits, which have not yet been defined for the insulin tolerance test (ITT). The study aim was to define the normative cortisol response to the ITT with the first- and second-generation Roche Elecsys^®Cortisol immunoassays (Elecsys^®Cort I and II).</p> Methods <p>Ninety participants underwent an ITT after an overnight fast. Blood samples were analysed by both Elecsys^®Cort I and II. The normative cortisol cut-off limit to the ITT was defined as the 2.5th percentile of the peak cortisol level for each assay. The percentage of healthy participants with peak cortisol below the cut-off limits defined for the ACTH test, 500 nmol/L for Elecsys^®Cort I and 420 nmol/L for Elecsys^®Cort II, respectively, was calculated as the false-positive rate.</p> Results <p>The mean peak cortisol was 608 ± 102 nmol/L with Elecsys^®Cort I and 501 ± 89 nmol/L with Elecsys^®Cort II. The 2.5th percentile was 404 nmol/L with Elecsys^®Cort I and 320 nmol/L with Elecsys^®Cort II; 5th percentiles were 451 nmol/L and 348 nmol/L, respectively. False-positive rates were 15% with Elecsys^®Cort I and 18% with Elecsys^®Cort II.</p> Conclusion <p>Elecsys^®Cort II demonstrated a lower normative cortisol response to the ITT compared with Elecsys^®Cort I, and the commonly used ACTH test thresholds. These data underline the need for assay- and test-specific cut-off limits to confirm or reject adrenal insufficiency.</p>