Testing for Cushing’s syndrome is a multi-step process that starts with screening tests to confirm excess cortisol, then moves to blood work and imaging to pinpoint the cause. No single test can diagnose Cushing’s on its own. Most endocrinologists use at least two different screening tests before making a diagnosis, because cortisol levels naturally fluctuate and several common medications can skew results.
Three First-Line Screening Tests
The Endocrine Society guidelines recommend starting with one of three main screening tests, and often repeating it or combining it with a second test to improve accuracy. Each measures cortisol differently, and your doctor will choose based on your specific situation.
24-Hour Urine Free Cortisol
This test measures the total amount of cortisol your body excretes over a full day. You collect all of your urine for 24 hours in a special container, starting the morning after your first bathroom trip (that first morning void gets flushed). Every drop after that goes into the container for the next 24 hours. Store the container in your refrigerator or at room temperature. Your doctor will typically ask you to do this collection at least twice, sometimes three times in a row, because a single collection can miss the excess cortisol on a “normal” day.
The upper limit of normal varies by sex: roughly 147 nmol/day for women and 238 nmol/day for men when measured by modern liquid chromatography methods. However, a result that’s only slightly above normal doesn’t clinch the diagnosis. Endocrinologists generally look for values at least double the upper reference range before they feel confident about a Cushing’s diagnosis.
This test is the preferred option during pregnancy, since the other screening tests involve medications that may not be safe. It’s less reliable in people with severe kidney disease, where urine output is compromised.
Late-Night Salivary Cortisol
In a healthy body, cortisol drops to its lowest point late at night. In Cushing’s syndrome, that nighttime dip disappears. To catch this, you collect a saliva sample between 10 p.m. and midnight using a small cotton swab or tube provided by your lab. You’ll do this on two separate nights.
A cortisol level above about 2.25 nmol/L in the saliva sample is considered a positive result, with roughly 92% sensitivity and 89% specificity. This test is convenient because you do it at home, and it’s a good option for people who find the 24-hour urine collection difficult to complete accurately. It’s also recommended alongside the dexamethasone suppression test for people who have an adrenal mass found incidentally on imaging.
Overnight Dexamethasone Suppression Test
Dexamethasone is a synthetic steroid. In a healthy person, taking a small dose suppresses the brain’s signal to produce cortisol. If cortisol stays high despite the medication, something is overriding the normal feedback loop.
You take a 1 mg dexamethasone tablet at 11 p.m. the night before, then have your blood drawn the following morning, usually around 8 a.m. A morning cortisol level above 1.8 mcg/dL (50 nmol/L) is considered a positive result. This cutoff is deliberately set low to avoid missing true cases, which means the test catches most people with Cushing’s but also flags some who don’t have it. The specificity is only 60 to 80%, so false positives are common and a positive result always needs confirmation with additional testing.
This is the preferred first test for people with severe kidney failure, where urine collection is unreliable.
What Can Cause False Results
Several common medications interfere with the dexamethasone suppression test and can produce a false positive. These include oral birth control pills, estrogen therapy, anti-seizure medications, antibiotics, diuretics, and any medication containing corticosteroids like prednisone or hydrocortisone. Your doctor may ask you to stop certain medications before testing, or choose a different screening method if stopping isn’t safe.
Physical and emotional stress, depression, alcoholism, obesity, and polycystic ovary syndrome can also raise cortisol enough to mimic Cushing’s syndrome on screening tests. These “pseudo-Cushing’s” states are one reason the diagnostic process requires multiple confirmatory steps rather than relying on a single abnormal result.
Finding the Source: ACTH-Dependent vs. ACTH-Independent
Once excess cortisol is confirmed, the next step is figuring out why it’s happening. The key measurement here is ACTH, a hormone produced by the pituitary gland that tells the adrenal glands to make cortisol. A simple blood draw, usually taken in the morning, reveals whether ACTH is driving the problem or not.
If ACTH is consistently below 5 pg/mL, the adrenal glands are producing cortisol on their own without being told to. This is called ACTH-independent Cushing’s, and it’s almost always caused by a tumor on one of the adrenal glands. The pituitary gland has essentially shut down its ACTH production because cortisol is already so high.
If ACTH is measurably elevated, something is producing too much of the hormone and overstimulating the adrenals. About 70% of the time, the culprit is a small, benign tumor on the pituitary gland (called Cushing’s disease specifically). Less commonly, a tumor somewhere else in the body, often in the lungs, produces ACTH on its own.
Telling Pituitary From Ectopic ACTH Sources
When ACTH levels are elevated, distinguishing a pituitary tumor from an ectopic source matters enormously because the treatments are completely different. Two tests help make this distinction.
The high-dose dexamethasone suppression test uses 8 mg of dexamethasone instead of 1 mg. Pituitary tumors usually retain some responsiveness to feedback, so cortisol will drop. A suppression of 69% or more from baseline strongly suggests a pituitary source, with about 90% specificity. Ectopic tumors, on the other hand, tend to ignore the dexamethasone entirely and keep pumping out ACTH.
The most definitive test is inferior petrosal sinus sampling. This is an invasive procedure performed by an interventional radiologist. Thin catheters are threaded through veins in the groin up to the small blood vessels that drain directly from the pituitary gland. Blood is drawn simultaneously from these pituitary-adjacent veins and from a vein in the arm. If the ACTH concentration near the pituitary is 1.3 times or more the level in the arm, the pituitary is confirmed as the source. Ratios of 0.7 or lower point to an ectopic source. This test has 95 to 99% sensitivity and over 90% diagnostic accuracy, making it the reference standard when other tests are inconclusive.
Imaging to Locate the Tumor
Imaging comes after the biochemical tests have narrowed down the source. Scanning before you know where to look can be misleading, since incidental, non-functioning tumors in the pituitary and adrenal glands are surprisingly common in the general population.
For suspected pituitary Cushing’s, a high-resolution MRI of the pituitary is performed with a contrast dye injection. The scan uses very thin slices (2 to 3 mm) and images taken at 10-second intervals to catch small tumors that enhance differently from normal pituitary tissue. Most pituitary tumors causing Cushing’s are microadenomas, smaller than 10 mm, with an average detected size of about 6 mm. Some are small enough that even a good MRI misses them, which is when petrosal sinus sampling becomes essential.
For suspected adrenal Cushing’s, a CT scan of the abdomen with thin slices (2.5 to 3 mm) and contrast is the standard approach. Adrenal adenomas typically appear as well-defined round nodules measuring 1 to 5 cm. Adrenal cancers tend to be much larger, averaging about 9 cm, and look more irregular on imaging. In rare forms of adrenal Cushing’s, the glands may contain multiple small nodules, sometimes smaller than 6 mm.
When an ectopic ACTH source is suspected, contrast-enhanced CT of the chest is usually the first scan, since the lungs are the most common location for ACTH-producing tumors outside the pituitary. CT or MRI of the abdomen follows if the chest scan is negative.
What the Testing Timeline Looks Like
From first suspicion to a definitive diagnosis, testing for Cushing’s syndrome often takes weeks to months. Initial screening requires at least two abnormal results from different tests or repeated collections. ACTH measurement and the high-dose dexamethasone test add another round of appointments and lab work. Imaging follows, and if results are ambiguous, petrosal sinus sampling may be scheduled, which requires a hospital visit.
This drawn-out process can feel frustrating, but it exists for good reason. Cortisol fluctuates naturally, many conditions mimic Cushing’s biochemically, and treatment (usually surgery) is significant enough that doctors want to be certain before proceeding. If your screening results are borderline, your endocrinologist may ask you to repeat testing after a few weeks or months rather than moving forward with an uncertain diagnosis.