Testing for Cushing’s syndrome involves a step-by-step process that starts with screening tests to measure your cortisol levels, then narrows down the cause if results come back abnormal. No single test can diagnose Cushing’s on its own. Most people need at least two different screening tests, followed by additional bloodwork and imaging if those results point toward excess cortisol production.
Who Should Be Tested
Cushing’s syndrome is not something doctors screen for routinely. The Endocrine Society recommends testing in specific situations: people who develop features unusual for their age (like osteoporosis or high blood pressure in a younger person), people with multiple progressive symptoms that fit the pattern (weight gain concentrated in the face and trunk, easy bruising, wide purple stretch marks, muscle weakness), and children whose growth is slowing while their weight is increasing. People with an adrenal mass found incidentally on imaging are also candidates for testing.
Before any biochemical testing, your doctor should review all medications you take. Steroid medications, including creams, inhalers, and joint injections, can cause the same symptoms and lab results as Cushing’s syndrome. Ruling out this external source of cortisol is the essential first step.
The Three First-Line Screening Tests
There are three tests considered reliable enough to use as initial screens. Your doctor will choose based on your specific situation, and you’ll typically need to do at least two of them.
24-Hour Urine Cortisol
This test measures the total amount of cortisol your body excretes over a full day. You collect all of your urine into a container for 24 hours, then submit it to a lab. Because cortisol levels naturally fluctuate throughout the day, capturing an entire day’s output gives a more complete picture than a single blood draw. Some doctors request two separate 24-hour collections on different days to improve accuracy.
Late-Night Salivary Cortisol
Cortisol normally drops to its lowest point late at night. In Cushing’s syndrome, this nighttime dip doesn’t happen. You collect a saliva sample at home between 11 p.m. and midnight using a small cotton swab or tube provided by the lab. A normal result is below 100 ng/dL. Confirmed Cushing’s patients typically have levels ranging from 100 ng/dL up to 6,000 ng/dL. You’ll usually be asked to collect samples on at least two separate nights.
A few things can falsely elevate your results. Smoking before collecting the sample, eating licorice (real licorice root, not the candy flavoring), and blood contamination from bleeding gums can all push levels higher than they truly are. Avoid these before collection.
Overnight Dexamethasone Suppression Test
This test checks whether your body responds normally to a signal that should shut down cortisol production. You take a 1 mg dexamethasone pill (a synthetic steroid) at 11 p.m., then have your blood drawn at 8 a.m. the next morning. In a healthy person, the dexamethasone tells the brain to stop stimulating cortisol, so morning levels drop. A fasting cortisol level below 1.8 mcg/dL is considered normal. If your cortisol stays above that threshold, the normal feedback loop isn’t working, which is a hallmark of Cushing’s.
Oral contraceptives and other estrogen-containing medications can interfere with this test. Estrogen raises the level of a protein in your blood that binds to cortisol, making total cortisol appear higher than it actually is. This can produce a false-positive result. If you take estrogen, your doctor may choose a different screening test or ask you to stop the medication several weeks beforehand.
What Happens After Screening
Two concordant positive results from different screening tests generally confirm that excess cortisol is present. On the other hand, if two different tests both come back normal, Cushing’s syndrome is effectively ruled out, with the rare exception of cyclical Cushing’s, where cortisol production fluctuates. If your results are mixed (one positive, one negative), your doctor will typically recommend further evaluation rather than stopping the workup.
Finding the Source of Excess Cortisol
Once excess cortisol is confirmed, the next step is figuring out where it’s coming from. The key test here measures ACTH, the hormone your pituitary gland releases to tell the adrenal glands to produce cortisol.
An ACTH level above 15 pg/mL suggests the problem is “ACTH-dependent,” meaning something is producing too much ACTH, which in turn drives cortisol production. This is most commonly a small pituitary tumor (Cushing’s disease) or, less often, a tumor elsewhere in the body. An ACTH level below 5 pg/mL points to an “ACTH-independent” cause, meaning the adrenal glands themselves are overproducing cortisol, usually due to an adrenal tumor. This distinction shapes every decision that follows.
Imaging to Locate the Problem
If ACTH levels point toward the pituitary gland, an MRI of the brain is the standard next step. MRI identifies about 73% of pituitary adenomas overall and roughly 71% of microadenomas, which are tumors smaller than 10 mm. That means nearly 3 in 10 small pituitary tumors won’t show up on MRI, which is why a normal scan doesn’t necessarily rule out a pituitary source.
If ACTH levels suggest an adrenal cause, imaging focuses on the adrenal glands, typically with a CT scan. For pregnant patients, ultrasound of the kidneys and adrenal area is preferred over CT to avoid radiation exposure.
When MRI Isn’t Enough: Sinus Sampling
When the ACTH level is high but MRI doesn’t clearly show a pituitary tumor, or when doctors need to confirm the pituitary is truly the source, a procedure called inferior petrosal sinus sampling (IPSS) can help. This is an invasive test performed by an interventional radiologist. Small catheters are threaded through veins in the groin up to the sinuses that drain blood directly from the pituitary gland. Blood samples are taken from these sinuses and compared to a sample from a peripheral vein.
If the ACTH concentration in the pituitary drainage is more than twice the level in peripheral blood, the pituitary is confirmed as the source. This ratio is measured both at baseline and after an injection of a stimulating agent to improve accuracy. IPSS is considered the gold standard for confirming pituitary Cushing’s disease when imaging is inconclusive.
Testing During Pregnancy
Pregnancy complicates Cushing’s testing because cortisol naturally rises during pregnancy, and some standard tests become unreliable. The Endocrine Society recommends using 24-hour urine cortisol and late-night salivary or serum cortisol for pregnant patients. The dexamethasone suppression test is generally avoided because pregnancy itself can alter how the body processes the drug. If a pituitary tumor is suspected, non-contrast MRI may be used, though its safety during pregnancy hasn’t been fully established.
What the Testing Process Looks Like Overall
For most people, the testing journey takes weeks to months. Initial screening involves one or two outpatient tests. If those are positive, confirmatory tests, ACTH measurement, and imaging follow. Some results require repeat testing because cortisol levels naturally vary, certain medications interfere, and some cases of Cushing’s produce cortisol intermittently rather than constantly. The process can feel slow, but each step narrows the diagnosis and ensures treatment targets the right cause.