What is the best way to test for cortisol levels?

Testing for Cortisol Levels

For screening hypercortisolism (Cushing's syndrome), start with late-night salivary cortisol (LNSC) collecting 2-3 samples at bedtime, or use the overnight 1-mg dexamethasone suppression test (DST) with cortisol measured at 8:00 AM; for evaluating adrenal insufficiency, obtain a single morning serum cortisol at 8:00-9:00 AM. 1, 2

For Suspected Cushing's Syndrome (Hypercortisolism)

First-Line Screening Tests

Choose based on patient circumstances:

• Late-Night Salivary Cortisol (LNSC): Collect at least 2-3 samples at the patient's usual bedtime (typically 11 PM-midnight) on consecutive days, with sensitivity of 95% and specificity of 93-100% 1, 3

  • This test detects loss of the normal circadian nadir of cortisol secretion that occurs in Cushing's syndrome 1
  • Critical contraindication: Never use LNSC in night-shift workers or anyone with disrupted sleep-wake cycles, as their cortisol peaks occur at different times 1, 4
  • Mass spectrometry is preferred over immunoassay to avoid contamination from topical hydrocortisone preparations 1

• Overnight 1-mg Dexamethasone Suppression Test (DST): Give 1 mg dexamethasone between 11 PM-midnight, measure serum cortisol at 8:00 AM the next morning 1

  • Normal response: cortisol <1.8 μg/dL (50 nmol/L) excludes dysregulated cortisol production 1
  • Values >5 μg/dL (138 nmol/L) indicate autonomous cortisol secretion with >90% sensitivity 2
  • Preferred for shift workers and patients with irregular sleep schedules 1, 4
  • Major pitfall: False positives occur in women taking oral estrogen-containing contraceptives due to increased cortisol-binding globulin 1
  • Consider measuring dexamethasone levels simultaneously with cortisol to identify false positives from malabsorption or drug interactions with CYP3A4 inducers (phenobarbital, carbamazepine, St. John's wort) 1, 2

• 24-Hour Urinary Free Cortisol (UFC): Collect at least 2-3 separate 24-hour urine collections to account for day-to-day variability of up to 50% 1, 3

  • Sensitivity 89%, specificity 100% 3
  • Advantage: Independent of cortisol-binding globulin changes and dexamethasone compliance 1
  • Avoid in patients with renal impairment (CrCl <60 mL/min) or significant polyuria (>5 L/24 hours), as glomerular filtration rate strongly affects results 1

Testing Algorithm for Cushing's Syndrome

1. Exclude iatrogenic causes: Review all glucocorticoid use (including topical, inhaled, and herbal preparations with steroid additives) before any testing 3

2. Initial screening: Perform 2-3 different first-line tests (LNSC, DST, and/or UFC) based on clinical suspicion and patient circumstances 1

3. If any test is abnormal: Repeat 1-2 screening tests to confirm before proceeding to localization studies 1, 3

4. For cyclic Cushing's syndrome: Use multiple sequential LNSC measurements over weeks to months, as patients alternate between normal and excess cortisol secretion 1, 3

For Suspected Adrenal Insufficiency

Morning Serum Cortisol

• Timing is critical: Draw blood at 8:00-9:00 AM to capture the physiologic peak of cortisol secretion 2

  • This timing provides optimal sensitivity (68%) and specificity (100%) for detecting adrenal dysfunction 2

• Interpretation thresholds (using nmol/L):

  • ≥300 nmol/L (10.9 μg/dL): Excludes adrenal insufficiency; no further testing needed 5, 6
  • <110 nmol/L (4.0 μg/dL): Strongly suggests adrenal insufficiency; proceed to ACTH stimulation test 5
  • 110-300 nmol/L: Indeterminate; requires ACTH stimulation test for definitive diagnosis 5, 6

• For afternoon samples (12 PM-6 PM): Use a lower threshold of <250 nmol/L (9.1 μg/dL) to maintain 96% sensitivity in outpatients 6

Simultaneous ACTH Measurement

• Always draw ACTH and cortisol together at 8:00-9:00 AM from the same blood draw to distinguish primary from secondary adrenal insufficiency 2
  • Primary adrenal insufficiency: ACTH elevated (>1.1 pmol/L or >5 ng/L) with low cortisol 2
  • Secondary (central) adrenal insufficiency: Both ACTH and cortisol low 2
  • Critical handling requirement: ACTH is extremely labile and requires immediate processing on ice; coordinate with laboratory before drawing 2

Common Pitfalls to Avoid

• Never interpret cortisol without knowing collection time: The same cortisol value may be normal at 9:00 AM but pathologically elevated at midnight 2

• Document medications affecting cortisol-binding globulin: Oral estrogens, pregnancy, and chronic active hepatitis increase total cortisol levels and cause false interpretation 1, 2

• Avoid physical/mental stress before testing: Recent strenuous exercise or cognitive assessments significantly elevate cortisol; patients should avoid exercise for 24-48 hours before blood draw 2

• Pseudo-Cushing's states: Severe obesity, uncontrolled diabetes, depression, and alcohol use disorder can cause mildly elevated results on all screening tests 1, 3

  • These patients typically have cortisol values only mildly above normal 1
  • Consider repeating tests after 3-6 months or treating the underlying condition 1

• Shift workers require special consideration: Standard morning cortisol and LNSC are unreliable; use DST instead 1, 4