Testing for adrenal insufficiency typically starts with a simple morning blood draw to measure cortisol, then moves to a stimulation test if results fall in a gray zone. The process is straightforward, but timing, medications, and the type of adrenal insufficiency suspected all influence which tests you’ll need and how to interpret them.
The Morning Cortisol Blood Test
Cortisol follows a daily rhythm, peaking between 6 and 8 a.m. and dropping to its lowest point around midnight. A morning blood draw takes advantage of that natural peak: if your cortisol is low even when it should be at its highest, that’s a strong signal something is off.
The thresholds are fairly clear-cut at the extremes. A morning cortisol below 3 μg/dL is consistent with adrenal insufficiency and often doesn’t require further testing if your symptoms match. On the other end, a value above 13 to 18 μg/dL (the exact cutoff depends on the lab’s assay) effectively rules it out. Values between those two numbers land in an indeterminate zone, which is where stimulation testing comes in.
For people tapering off steroid medications like prednisone, the Endocrine Society recommends morning cortisol as the first check of whether the adrenal glands have woken back up. A level above 10 μg/dL (300 nmol/L) suggests the body’s stress-hormone axis has recovered. If it falls below that, you’ll likely stay on a low replacement dose and retest weeks to months later.
How to Prepare for a Cortisol Test
Blood samples are usually drawn twice on the same day: once in the early morning and again around 4 p.m., when levels are normally much lower. You don’t necessarily need to fast, but you may be asked to rest quietly before the draw because both stress and exercise can temporarily spike cortisol and skew the results.
Several medications can interfere with cortisol measurements. Estrogen (including oral contraceptives), spironolactone, hydrocortisone, and cortisone can all push baseline cortisol readings artificially high by increasing the binding proteins in your blood. Let the ordering clinician know about every medication and supplement you take so they can decide whether anything needs to be paused beforehand. If a saliva cortisol test is used instead, you’ll need to avoid eating, drinking, brushing, or flossing for at least 30 minutes before the sample.
The ACTH Stimulation Test
When a morning cortisol falls in that gray zone, the next step is usually the ACTH stimulation test, often considered the gold standard for confirming adrenal insufficiency. ACTH is the pituitary hormone that tells the adrenal glands to produce cortisol. In this test, a synthetic version of ACTH is injected, and your cortisol is measured at baseline, then again at 30 and 60 minutes.
Healthy adrenal glands respond with a robust cortisol rise, typically peaking above 18 μg/dL. If the glands fail to reach that threshold, adrenal insufficiency is confirmed. The test takes about an hour total, is done as an outpatient procedure, and is generally well tolerated. You may be asked to temporarily stop hydrocortisone or other glucocorticoids before the test because they can mask an abnormal result.
One limitation: the ACTH stimulation test is best at detecting primary adrenal insufficiency (Addison’s disease), where the adrenal glands themselves are damaged. It can sometimes miss early or partial cases of secondary adrenal insufficiency, where the problem originates in the pituitary gland, because the adrenal glands haven’t had time to fully shrink from disuse.
Testing for Pituitary-Related Adrenal Insufficiency
When the suspicion is that the pituitary gland or hypothalamus is the source of the problem (secondary or tertiary adrenal insufficiency), more specialized tests may be needed.
Insulin Tolerance Test
The insulin tolerance test is considered the most definitive way to evaluate the entire stress-hormone chain from brain to adrenal gland. Insulin is injected to deliberately lower blood sugar, which is a powerful stress signal. The body should respond by ramping up cortisol production. For the test to be valid, blood glucose must drop below about 40 mg/dL (2.2 mmol/L) or the patient must show clear symptoms of low blood sugar like sweating, shakiness, or a rapid heartbeat.
Because it involves intentional hypoglycemia, this test carries real risks and isn’t appropriate for everyone. It’s contraindicated in people over 60, those with heart disease or epilepsy, and anyone with severely low baseline cortisol. It requires continuous monitoring in a clinical setting with emergency glucose available. For these reasons, it’s used selectively rather than as a routine screening tool.
Glucagon Stimulation Test
When the insulin tolerance test is too risky, the glucagon stimulation test offers a safer alternative. Glucagon is injected to indirectly trigger a cortisol response. A study published in the Journal of Clinical Endocrinology & Metabolism found that using a peak cortisol cutoff of 14.6 μg/dL after glucagon, the test achieved 100% specificity (meaning it didn’t falsely diagnose anyone) with a sensitivity of about 67%. That lower sensitivity means it catches roughly two-thirds of true cases, so a normal result is very reassuring, but an abnormal result may still need confirmation.
CRH Stimulation Test
A CRH stimulation test can help distinguish between secondary adrenal insufficiency (pituitary origin) and tertiary adrenal insufficiency (hypothalamic origin). CRH is the brain hormone that tells the pituitary to release ACTH. When CRH is injected, someone with a hypothalamic problem will show rising ACTH levels because their pituitary is intact and responsive. Someone with a damaged pituitary will show flat ACTH levels. This distinction matters for treatment planning, though CRH has faced supply shortages in recent years, limiting its availability.
Identifying the Underlying Cause
Once adrenal insufficiency is confirmed, the next question is why. The answer depends on where the problem sits.
For primary adrenal insufficiency, the most common cause in developed countries is autoimmune destruction of the adrenal glands. A blood test for 21-hydroxylase antibodies can confirm this. These antibodies are present in up to 90% of people with autoimmune Addison’s disease, and the test has a clinical sensitivity of about 87% with a specificity above 99%. A positive result essentially confirms an autoimmune cause. If antibodies are negative, imaging of the adrenal glands (usually a CT scan) is typically ordered to look for other causes like infection, bleeding, or infiltrative disease.
For secondary adrenal insufficiency, ACTH levels will be low or inappropriately normal despite low cortisol. The most common cause by far is prolonged use of glucocorticoid medications, which suppress the pituitary’s ability to produce ACTH. If steroid use isn’t the explanation, an MRI of the pituitary gland is usually the next step to check for tumors, inflammation, or other structural problems.
What ACTH Levels Tell You
Measuring ACTH alongside cortisol is one of the simplest ways to distinguish primary from secondary adrenal insufficiency. In primary disease, the adrenal glands are failing, so the pituitary compensates by producing excess ACTH. You’ll see low cortisol paired with high ACTH. In secondary disease, the pituitary itself is the problem, so both ACTH and cortisol are low. This pairing is typically checked during the initial workup and helps direct subsequent testing.
Testing After Steroid Use
If you’ve been on glucocorticoid medications (prednisone, dexamethasone, or similar) for weeks or longer, your adrenal glands may have gone dormant. The Endocrine Society advises against routinely ordering stimulation tests during a taper. Instead, a simple morning cortisol check is preferred. If it’s above 10 μg/dL, your system is likely recovering. If not, you continue a low physiologic dose and retest. This process can take weeks to months, and occasionally longer for people who were on high doses for extended periods.
Routine testing is also not recommended while you’re still on doses above the body’s normal daily cortisol production (roughly equivalent to 5 mg of prednisone per day) or while you still need the medication for the condition it was prescribed for. Testing at those points would simply confirm suppression that’s expected and doesn’t change management.