What is the optimal time of day to perform a cosyntropin (adrenocorticotropic hormone, ACTH) stimulation test?

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Last updated: June 17, 2025View editorial policy

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From the Guidelines

The cosyntropin stimulation test should ideally be performed in the morning, between 8 AM and 9 AM, to align with the natural diurnal rhythm of cortisol and ensure the most reliable baseline cortisol values for comparison. This recommendation is based on the understanding of cortisol's natural fluctuation throughout the day, which is not directly addressed in the provided evidence 1. However, the evidence does discuss the high-dose and low-dose ACTH stimulation tests, highlighting their diagnostic accuracy for adrenal insufficiency 1.

When performing the test, a baseline cortisol level is drawn, followed by the administration of synthetic ACTH (cosyntropin), typically 250 mcg, intravenously or intramuscularly. Additional cortisol measurements are then taken at 30 and 60 minutes post-administration. The morning timing is crucial because it provides a more accurate baseline, as cortisol levels naturally decline throughout the day. This could lead to misinterpretation if the test is conducted in the afternoon or evening.

Key considerations for the test include:

  • Documenting the time of the test for proper interpretation
  • Avoiding exogenous glucocorticoids before the test when possible to prevent interference with results
  • Conducting the test after an overnight fast, with water permitted
  • Understanding that while the test can be performed at any time of day if necessary, morning results are more reliable due to the natural cortisol rhythm. The provided evidence 1 focuses on the comparison between high-dose and low-dose ACTH tests rather than the timing, but clinical practice aligns with morning testing for optimal results.

From the Research

Optimal Time of Day for Cosyntropin Stim Test

The optimal time of day to perform a cosyntropin stimulation test is not strictly defined, but research suggests that the test can be performed at various times of the day with comparable results.

  • A study published in 2019 2 found that using a short plastic tube, the low-dose cosyntropin test provided comparable morning and afternoon 30-minute stimulated serum cortisol and salivary free cortisol concentrations in healthy volunteers.
  • However, the same study noted that the mean baseline morning serum cortisol concentration was higher in the morning than in the afternoon, while the mean absolute serum cortisol concentration increment between baseline and 30-minute time point was higher in the afternoon than in the morning.

Cortisol Response

The cortisol response to cosyntropin stimulation can vary depending on the time of day and the dose of cosyntropin used.

  • A study published in 2008 3 suggested that the 1-microg cosyntropin stimulation test is more sensitive than the 250-microg test for diagnosing secondary adrenal insufficiency in nonstressed patients.
  • Another study published in 2017 4 compared the 1 μg, 25 μg, and 250 μg cosyntropin stimulation tests and found that all tests had excellent correlations with the insulin tolerance test when appropriate cut-offs were used.
  • A 2022 study 5 evaluated the proper cortisol values during the standard-dose cosyntropin stimulation test using the Elecsys Cortisol II assay and found that the lowest cortisol levels at 30 and 60 minutes after cosyntropin administration were 15.7 and 17.9 µg/dL, respectively.

Administration Route

The route of administration of cosyntropin can also affect the cortisol response.

  • A study published in 1992 6 compared the intravenous and intramuscular routes of administering cosyntropin in cats and found that intravenous administration induced significantly greater and more prolonged adrenocortical stimulation than intramuscular administration.

Professional Medical Disclaimer

This information is intended for healthcare professionals. Any medical decision-making should rely on clinical judgment and independently verified information. The content provided herein does not replace professional discretion and should be considered supplementary to established clinical guidelines. Healthcare providers should verify all information against primary literature and current practice standards before application in patient care. Dr.Oracle assumes no liability for clinical decisions based on this content.

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