24-hour urine cortisol test: how to collect it, what it shows and its limits

A 24-hour urine cortisol test, usually called a 24-hour urinary free cortisol (UFC) test, measures all the free cortisol your kidneys pass into your urine over a full day and night. Free cortisol is the unbound, active form. Because the collection spans 24 hours, it takes in the whole daily rhythm and the smaller pulses that make a single blood sample so dependent on timing.¹ Doctors use it mainly as one of the first tests for Cushing's syndrome, usually asking for at least two collections.² It is not difficult, but it is fiddly, and the result is only as good as the collection.

What the test measures

Most blood tests measure total cortisol, most of which is bound to carrier proteins. A urine test measures free cortisol instead, so, unlike a blood test, it is not affected by changes in the main carrier protein, corticosteroid-binding globulin (CBG).¹ The Endocrine Society guideline notes that oral oestrogen, which raises CBG and so pushes up total blood cortisol, does not affect urinary free cortisol in the same way.²

One collection gives one day's total. It includes that day's morning high and night-time low,¹ but says nothing about the day before or the day after. Many laboratories also measure creatinine, a waste product passed in urine at a fairly steady rate, in the same sample, which helps show whether the collection looks complete.

How to collect a 24-hour urine sample

The laboratory gives you a large container and its own instructions, which you should follow exactly.³ The usual method is as follows.⁴

1. On day 1, pass your first urine of the morning into the toilet as normal. This starts the 24 hours, so note the time.
2. Collect all the urine you pass for the rest of the day and night in the container.
3. On day 2, pass your first urine on getting up into the container. This ends the collection.
4. Keep the container capped and in the fridge or a cool place throughout. Label it with your name, the date and the time you finished, and return it as instructed.

Plan the day. Pick one when you can stay near home or take the container with you, because missing a single pass of urine, or adding one from outside the 24 hours, makes the total wrong. If you do miss one, tell the laboratory or the doctor who asked for the test; they may ask you to start again on another day.

You may be asked to avoid vigorous exercise the day before, and to stop certain medicines for a while, including anti-seizure medicines, oestrogen, synthetic glucocorticoids such as prednisolone, and androgens.³ Do not stop any medicine without your doctor's advice. Drink as you normally would unless told otherwise, because the volume of urine itself influences the result.⁵

In the UK the test is usually arranged by a GP or a specialist; a GP can arrange cortisol tests of blood, urine or saliva.⁶ In the US it can also be ordered privately and collected through a partner centre, as in the example below.

Normal ranges and what results mean

Ranges depend on the laboratory and its method, so the range on your report is the one that counts.³ Two examples show the scale.

A result above the range can reflect Cushing's syndrome, an adrenal tumour, a tumour elsewhere making the hormone ACTH, severe depression or severe stress. A result below it can reflect Addison's disease, a pituitary problem or steroid medicines.³ A low urine result is not how adrenal insufficiency is diagnosed, though: that relies on blood tests, the standard being the short Synacthen test.⁸

How doctors use it to test for Cushing's syndrome

The Endocrine Society guideline lists four first-line tests for Cushing's syndrome: 24-hour urinary free cortisol (at least two collections), late-night salivary cortisol (two samples), the 1 mg overnight dexamethasone suppression test, and a longer 48-hour dexamethasone test.² The 2021 Pituitary Society consensus recommends starting with the dexamethasone test, urine and/or late-night saliva, and advises two or three urine collections to capture variability.⁵ It states that all the standard tests have a sensitivity above 90 per cent, with lower specificity: they rarely miss the condition but can flag people who turn out not to have it.⁵ Late-night saliva and urine perform similarly.²

Urine has a particular role when Cushing's may be cyclic, with bursts of excess cortisol separated by normal spells. The Endocrine Society suggests urine or late-night saliva rather than dexamethasone tests in that situation, but warns that a urine result can be normal if it is collected while the disease is quiet.² After diagnosis, urine and late-night saliva are used to monitor medical treatment.⁵ See testing for Cushing's syndrome and the dexamethasone suppression test.

Why one collection is not enough

Even a whole day's urine varies a great deal from one day to the next. In 152 people with Cushing's disease, who provided more than 600 samples between them, results varied by 52 per cent on average within the same patient.⁷ Collecting more than two samples did not meaningfully reduce that variability, and the level did not track how severe the disease was.⁷ The 2021 consensus describes random variability in urinary free cortisol of up to 50 per cent.⁵ MedlinePlus notes that the test may need repeating three or more times over a few weeks to months.³

Healthy people vary too. In a month-long study of 17 healthy adults, weekly 24-hour urine results correlated at only r = 0.59 to 0.68 from one week to the next.⁹ A single collection is one day's reading, not your usual level.

What else affects the result

• Kidney function. The result can be falsely low when kidney function is reduced, with a creatinine clearance below 60 mL/min, and it falls further as kidney failure worsens.²
• Body and lifestyle. Sex, body mass index, age, urine volume and salt intake all influence urinary free cortisol.⁵
• Other conditions. Depression, anxiety disorders, poorly controlled diabetes and alcohol dependence can raise cortisol enough to give an abnormal result without Cushing's syndrome, and severe obesity can raise urinary free cortisol.²
• Medicines. Steroid medicines, oestrogen, anti-seizure medicines and androgens can all affect the test.³
• An incomplete collection. A missed pass of urine lowers the total, and one from outside the 24 hours raises it.

Urine or hair for long-term cortisol?

A 24-hour collection captures a whole day far better than a single blood sample, but it still captures only one day. Standard 24-hour urine and late-night saliva tests reflect at most 24 hours, which is why they can be normal in cyclic Cushing's syndrome.¹⁰ If your question is about the past few months, a hair test answers it more directly. Scalp hair grows about 1 cm a month and takes up cortisol as it grows,¹¹ and in the month-long study the same people's hair cortisol correlated at r = 0.84 from one month to the next, against 0.59 to 0.68 week to week for urine.⁹

The two do not measure exactly the same thing. In small studies, hair cortisol agreed only weakly with urine averaged over one to two months (r = 0.30, and Kendall's tau = 0.42), and neither result was statistically significant.⁹¹² Hair is also not part of the diagnostic guidelines for Cushing's syndrome, which use urine, saliva and dexamethasone tests.²⁵ For a long-term average it is the more consistent single-sample measure; for diagnosis, urine remains the test. See hair vs urine cortisol tests.