Cortisol Testing: Serum, Saliva and Urine Compared

Cortisol is tested in 3 very different ways, serum, saliva and urine, and for very different reasons. Clinical endocrinology uses cortisol tests to diagnose 2 important conditions: adrenal insufficiency and Cushing syndrome. Functional and wellness practice has used cortisol profiles, particularly salivary tests, to assess stress and diagnose adrenal fatigue. Understanding which test answers which question, and what affects the results, is essential.

2 clinical questions

Testing for adrenal insufficiency

Cortisol normally peaks in the early morning, so a serum sample taken around 8 to 9 am is the best screen. A very low morning cortisol makes adrenal insufficiency likely; a clearly high value makes it unlikely. Intermediate results require dynamic testing. Exact thresholds depend on the assay used, and laboratories provide their own cut offs, particularly as newer assays give lower values than older ones.

The ACTH stimulation test, in which synthetic ACTH is injected and cortisol measured afterwards, assesses the adrenal glands’ capacity to respond. A normal peak response excludes primary adrenal insufficiency. In recent onset secondary insufficiency, such as after pituitary surgery, the test may be normal initially because the adrenal glands have not yet atrophied, so clinical judgement and specialist input are needed.

Plasma ACTH, measured alongside morning cortisol, helps distinguish primary adrenal insufficiency, in which ACTH is high, from secondary, in which it is low or normal. Renin and aldosterone assess mineralocorticoid function in primary insufficiency.

Suspected adrenal crisis, with vomiting, collapse, low blood pressure or low sodium, is a medical emergency, and treatment must not wait for test results.

Testing for Cushing syndrome

Endocrine Society guidance recommends one or more of 3 screening tests, chosen according to the clinical situation.

• Late night salivary cortisol: in healthy people, cortisol reaches its lowest point around midnight. Loss of this nadir is an early feature of Cushing syndrome. 2 samples on separate nights are usually recommended. Shift work, disrupted sleep and contamination with topical hydrocortisone can affect results.
• Overnight dexamethasone suppression test: a small dose of dexamethasone taken late in the evening normally suppresses morning cortisol. Failure to suppress suggests Cushing syndrome. Medicines that speed dexamethasone metabolism, such as some antiepileptics, and oral oestrogen, which raises cortisol binding globulin, can cause false positive results.
• 24 hour urinary free cortisol: measures cortisol excreted over a day. At least 2 collections are usually recommended. Incomplete collections, high fluid intake and kidney impairment affect results.

Abnormal screening results are confirmed with further testing by an endocrinologist, who then determines the cause.

What affects cortisol results

A careful medication history is essential. Many patients do not consider inhaled or topical steroids, or steroid injections for joints, to be relevant, yet these can suppress the adrenal axis and affect results.

Salivary diurnal profiles and adrenal fatigue

Salivary cortisol profiles, typically 4 or more samples through the day, are used in some practice to diagnose adrenal fatigue and guide supplements. These profiles are sensitive to the exact timing of samples relative to waking, vary considerably from day to day in the same person and lack validated reference patterns for diagnosing any condition other than the loss of the late night nadir in Cushing syndrome. Systematic review has found no consistent evidence for adrenal fatigue as a condition. Such profiles are best regarded as research tools.

Hair cortisol

Hair cortisol reflects cortisol exposure over weeks to months and is increasingly used in research on chronic stress. It is not a validated clinical diagnostic test, although it has been explored as an adjunct in Cushing syndrome.

Worked cases

The pill and the high cortisol

A 28 year old woman with weight gain has a high morning serum cortisol. She takes a combined oral contraceptive, which raises cortisol binding globulin. Her doctor arranges late night salivary cortisol tests instead, which are normal.

The inhaled steroid

A 60 year old man with fatigue and dizziness on standing has a low morning cortisol. He has used high dose inhaled steroids for asthma for years and recently had a steroid injection in his knee. Secondary adrenal insufficiency from steroid use is suspected, and he is referred to endocrinology for assessment and a plan, including advice on steroid cover during illness.

The depressed patient

A 45 year old woman with severe depression and weight gain has a mildly abnormal overnight dexamethasone suppression test. Depression can produce this pattern. Further testing by an endocrinologist, alongside treatment of her depression, shows no Cushing syndrome.

The adrenal fatigue diagnosis

A 36 year old man brings a 4 point salivary profile labelled as showing adrenal fatigue. He is tired and stressed. The practitioner explains the limits of the test, reviews his sleep and workload, and his doctor checks for anaemia, thyroid disease and depression. No adrenal testing is indicated. His plan focuses on sleep and stress management.

Matching test to question

Cortisol tests are powerful when used to answer the right question with the right method: morning serum and stimulation testing for adrenal insufficiency, late night saliva, dexamethasone suppression or urinary cortisol for Cushing syndrome. Understanding the factors that alter results, and avoiding unvalidated profiles, prevents both missed diagnoses and false ones.