Is an Adrenocorticotropic hormone (ACTH) stimulation test required to be performed in the morning?

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Last updated: April 17, 2025View editorial policy

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From the Guidelines

An ACTH stimulation test does not have to be done in the morning, as it measures the adrenal glands' response to synthetic ACTH rather than relying on natural diurnal cortisol patterns. The test can be performed at any time of day, as it evaluates the adrenal glands' maximum capacity to produce cortisol in response to stimulation, making the time of day irrelevant 1. According to the study, recommended tests for diagnostic confirmation of hypophysitis, including adrenal function tests such as the ACTH stimulation test, are preferably conducted in the morning around 8 am, but this is not a strict requirement 1.

Some key points to consider when performing an ACTH stimulation test include:

  • A baseline cortisol level is drawn before administering 250 mcg of cosyntropin intravenously or intramuscularly
  • Cortisol measurements are taken at 30 and 60 minutes after injection
  • The test evaluates the adrenal glands' maximum capacity to produce cortisol in response to stimulation
  • Patients should follow any specific preparation instructions from their healthcare provider to ensure accurate results 1.

It's worth noting that while the time of day may not be critical for the ACTH stimulation test, some endocrinologists may still prefer morning testing for consistency in protocol or to obtain a morning baseline cortisol level simultaneously 1.

From the FDA Drug Label

The test may be performed at any time during the day but because of the physiological diurnal variation of plasma cortisol the criteria listed by Wood cannot apply. Key Points:

  • The ACTH stimulation test can be performed at any time during the day.
  • The physiological diurnal variation of plasma cortisol should be considered when interpreting the results. The ACTH stimulation test does not have to be done in the morning 2.

From the Research

ACTH Stimulation Test Timing

  • The ACTH stimulation test does not necessarily have to be done in the morning, as studies have shown that the test results are not significantly affected by the time of day 3, 4.
  • One study found that afternoon cortisol levels in response to ACTH were higher than morning levels at 5 and 15 minutes, but this difference was no longer notable at 30 minutes 3.
  • Another study found no differences in mean cortisol levels at 30 and 60 minutes after ACTH administration, regardless of the time of day the test was performed 4.

Test Interpretation

  • The 30-minute cortisol response to ACTH is considered a reliable criterion for measuring adrenal response, and it is not affected by basal cortisol level or time of day 3.
  • However, using a 30-minute cortisol level in isolation may result in false positive diagnoses of adrenal insufficiency, and a 60-minute value may be more reliable 4.
  • The interpretation of the ACTH stimulation test results should take into account the clinical presentation and other technical factors that can affect cortisol values and diagnostic accuracy 5.

Diagnostic Cutoffs

  • Different studies have proposed various diagnostic cutoffs for the ACTH stimulation test, depending on the assay used and other factors 6, 7.
  • For example, one study recommended a threshold of 14.6 μg/dL for the level of cortisol at 60 minutes after ACTH stimulation using the Abbott Architect immunoassay 6.
  • Another study suggested that an inadequate response of serum cortisol at 60 minutes to the ACTH stimulation test may be indicative of adrenal insufficiency in patients receiving high-dose glucocorticoid therapy 7.

References

Guideline

Guideline Directed Topic Overview

Dr.Oracle Medical Advisory Board & Editors, 2025

Research

New Diagnostic Cutoffs for Adrenal Insufficiency After Cosyntropin Stimulation Using Abbott Architect Cortisol Immunoassay.

Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 2022

Research

Adrenocorticotropic hormone stimulation test during high-dose glucocorticoid therapy.

Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists, 2009

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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