How do you test for morning cortisol levels?

How to Test for Morning Cortisol

Morning cortisol is tested using a blood draw (serum sample), not a urine test. 1

Blood Sample Collection Method

The standard approach is a venous blood draw performed between 8:00-9:00 AM to capture the physiologic peak of cortisol secretion. 1, 2 This timing is critical because:

• The 8:00-9:00 AM window provides optimal sensitivity (68%) and specificity (100%) for detecting ACTH-dependent hypercortisolism 2
• This timing serves as the standard reference point used in all diagnostic algorithms, including interpretation of overnight dexamethasone suppression tests 1
• The same cortisol value may be normal at 9:00 AM but pathologically elevated at midnight, making timing essential for accurate interpretation 1

Pre-Collection Patient Preparation

Patients must follow specific restrictions before blood collection to avoid falsely elevated results: 1

• No strenuous physical activity for 24-48 hours before the blood draw, as exercise significantly elevates cortisol levels 1
• Avoid cognitive assessments or mental stress immediately before the blood draw, as acute psychological stress increases cortisol secretion 1
• No smoking, vaping, or nicotine products for 4 hours before sampling 1
• No alcohol consumption for 12 hours before sampling 1
• Avoid teeth brushing or dental work within 1-2 hours of blood collection, as blood contamination can falsely elevate biomarker levels 1

Critical Medication History

Always document oral contraceptives, estrogen-containing medications, and pregnancy status before interpreting results. 1 These factors increase cortisol-binding globulin (CBG) levels, which can raise total cortisol measurements dramatically while free cortisol remains normal, leading to false interpretation of hypercortisolism 3, 1

Diagnostic Interpretation Thresholds

For morning serum cortisol (8:00-9:00 AM): 3, 1

• Normal range: 5-23 μg/dL (138-635 nmol/L), with most values between 10-20 μg/dL (276-552 nmol/L) 3, 1
• >14 μg/dL (>386 nmol/L): Effectively rules out adrenal insufficiency 3, 1
• <10 μg/dL (<276 nmol/L): Suggests possible adrenal insufficiency and warrants further testing 3
• <250 nmol/L with elevated ACTH in acute illness: Diagnostic of primary adrenal insufficiency 1

When ACTH Measurement is Needed

For distinguishing primary from secondary adrenal insufficiency, both ACTH and cortisol must be drawn simultaneously at 8:00-9:00 AM from the same blood draw. 2 ACTH is extremely labile and requires immediate processing on ice, so coordinate with the laboratory before drawing 2

Common Pitfalls to Avoid

• Never interpret a single cortisol value without knowing the exact time of collection 1
• Shift workers and patients with disrupted circadian rhythms should not have standard AM cortisol testing, as their cortisol peaks occur at different times and will yield unreliable results 2
• Treatment of suspected acute adrenal insufficiency should never be delayed by diagnostic procedures 1

Alternative: Urine Testing (Not for Morning Cortisol Specifically)

While morning cortisol itself is measured via blood, 24-hour urinary free cortisol (UFC) is a separate test used for evaluating hypercortisolism/Cushing's syndrome, not for assessing morning cortisol levels. 3 The 24-hour UFC measures total cortisol excretion over an entire day and requires complete collection with volume and creatinine measurement to assess completeness 3

A novel research approach using morning spot urinary cortisol-to-creatinine ratio (UCCR) has been proposed 4, but this is not yet standard practice and the established guidelines recommend serum blood testing for morning cortisol assessment 1, 2