At what time should an adrenocorticotropic hormone (ACTH) stimulation test, also known as an AM cortisol level, be drawn?

Morning Cortisol Testing Time Requirements

AM cortisol levels should be drawn between 7-9 AM to accurately reflect the normal diurnal peak of cortisol secretion. 1, 2

Timing Rationale and Importance

Morning cortisol testing is a critical component in evaluating adrenal function, particularly when assessing for adrenal insufficiency. The timing is essential because:

• Cortisol follows a diurnal rhythm with peak levels occurring in the early morning (7-9 AM)
• Samples collected outside this window may lead to falsely low results and misdiagnosis
• Guidelines specifically refer to "9 AM cortisol" when evaluating adrenal function 1

Specific Testing Protocols

For Suspected Adrenal Insufficiency:

• Draw baseline cortisol between 7-9 AM 2
• Values <108-140 nmol/L (<3.9-5.1 μg/dL) are highly suggestive of adrenal insufficiency 3
• Values >300-375 nmol/L (>10.8-13.6 μg/dL) typically exclude adrenal insufficiency 4, 5
• Values between these thresholds require confirmatory testing with an ACTH stimulation test

For ACTH Stimulation Testing:

1. Obtain baseline cortisol at 7-9 AM 6
2. Administer synthetic ACTH (cosyntropin):
   • Adults: 0.25 mg IV or IM 6
   • Children: 0.125 mg (birth to <2 years) or 0.25 mg (2-17 years) 6
3. Collect blood samples at baseline, 30 minutes, and 60 minutes after administration 6

Special Considerations

• Medication Interference: Stop glucocorticoids and spironolactone on the day of testing 6
• Long-acting glucocorticoids: Require longer washout period before testing 6
• Central vs. Primary Adrenal Insufficiency: Morning cortisol is useful for both but interpretation differs
• Hypophysitis Evaluation: 9 AM cortisol is part of the standard pituitary axis assessment 1

Clinical Interpretation

• If 9 AM cortisol <250 nmol/L or random cortisol <150 nmol/L with symptoms, hydrocortisone replacement is indicated 1
• For patients with suspected Cushing syndrome, morning cortisol is part of the diagnostic algorithm but must be interpreted alongside other tests 1
• The 30-minute cortisol response to ACTH is consistent regardless of basal cortisol level or time of day 7

Common Pitfalls to Avoid

1. Collecting samples outside the 7-9 AM window
2. Testing patients on exogenous glucocorticoids without proper washout
3. Relying solely on morning cortisol without considering clinical context
4. Failing to recognize that stress (illness, trauma) can falsely elevate cortisol levels
5. Not checking cortisol before initiating thyroid hormone replacement in patients with central hypothyroidism 2

Morning cortisol testing is a valuable first-line test for adrenal function assessment, but timing is critical for accurate interpretation and subsequent management decisions.