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Background The ACTH stimulation has low sensitivity for the diagnosis of hypercortisolism possibly as a result of biological and analytical variability. Hypothesis/Objectives To report the components of biological and analytical variability in serum cortisol concentration post- ACTH stimulation ([cortisol]) in healthy dogs. Animals Fourteen healthy harrier hound dogs. Methods The data were extracted from a separate, prospective, randomized, double-blinded, controlled discovery study in which dogs treated with vehicle control and 4 different doses of cortisone acetate ( CA) for 7 days had an ACTH stimulation test performed to confirm the dose-dependent effect of CA. The index of individuality (IoI), the critical difference between sequential measurements ( C D), and the number of measurements required to assess the homeostatic set point ( HSP) of [cortisol] with confidence intervals ( CI) of 90 and 95% were estimated. Results The IoI was equal to 1.1 and the C D was 3.3 μg/ dL (92 nmol/L). The number of measurements required to assess the HSP of [cortisol] with CI of 90 and 95% were 3 and 15, respectively. Additionally, mean [cortisol] was higher in males than in females (13.3 ± 4 μg/ dL [366 ± 114 nmol/L] vs. 11.5 ± 2.5 μg/ dL [318 ± 65 nmol/L], respectively; P = .046). As expected, treatment with CA resulted in a dose-dependent suppression of [cortisol]. Conclusions and Clinical Importance False-negative test results in hypercortisolism could occur when [cortisol] is outside of the individual's HSP and within the reference interval. The large C D emphasizes the importance of assessing clinically relevant parameters in the diagnosis and monitoring of HC. [ABSTRACT FROM AUTHOR]