The ACTH stimulation test is a blood test that measures how well your adrenal glands produce cortisol in response to an injected dose of synthetic adrenocorticotropic hormone (ACTH). It is the most widely used clinical tool for diagnosing adrenal insufficiency, a condition in which the adrenal glands do not make enough cortisol on their own. The test is straightforward, generally safe, and takes about an hour, but interpreting the results involves more nuance than most patients realize, and the version of the test your doctor chooses, the lab method used to measure cortisol, and even which medications you take can all shift what “normal” looks like.
What Happens During the Test
A nurse or technician draws a baseline blood sample, then injects a synthetic form of ACTH called cosyntropin (also known as tetracosactide outside the United States). This synthetic molecule contains only the first 24 amino acids of the full 39-amino-acid ACTH hormone, which is enough to trigger cortisol release while causing far fewer allergic reactions than the older animal-derived preparations that were used before the 1970s. Blood is drawn again at 30 minutes and sometimes at 60 minutes. The lab measures cortisol in each sample, and the peak value is compared against a cutoff to determine whether the adrenal response is adequate.
In a healthy person, injecting cosyntropin causes a brisk rise in cortisol. If the adrenal glands are damaged or if they have been understimulated for so long that they have atrophied, that cortisol rise will be blunted or absent. The traditional cutoff for a “pass” has been a peak cortisol somewhere around 500 to 550 nmol/L (roughly 18 to 20 µg/dL), though the exact number depends on which lab assay is used, a point that matters more than most clinicians historically appreciated.
High-Dose Versus Low-Dose Protocols
The standard high-dose version uses 250 micrograms of cosyntropin. That is a massive pharmacological dose, far more ACTH than the body would ever produce naturally. A low-dose version uses just 1 microgram. Both have been studied extensively, and a systematic review and meta-analysis published in the Journal of Clinical Endocrinology & Metabolism found that the two doses had similar overall diagnostic accuracy, with comparable sensitivity and specificity across adults and children.1The Journal of Clinical Endocrinology & Metabolism. ACTH Stimulation Tests for the Diagnosis of Adrenal Insufficiency: Systematic Review and Meta-Analysis A head-to-head comparison in patients with pituitary disease likewise found nearly identical diagnostic sensitivity between the two doses.2The Journal of Clinical Endocrinology & Metabolism. Comparison of Low and High Dose Corticotropin Stimulation Tests in Patients with Pituitary Disease
So why does the debate persist? The 250-microgram dose floods the adrenal glands with so much stimulation that mildly impaired glands can sometimes still produce a “normal” cortisol response, masking early or partial insufficiency. One study found that the 1-microgram test identified impaired adrenal function in about 95% of affected patients, while the high-dose version missed most of them at the same cortisol cutoff.3PubMed. Low-dose (1 microgram) adrenocorticotrophin (ACTH) stimulation as a screening test for impaired hypothalamo-pituitary-adrenal axis function: sensitivity, specificity and accuracy in comparison with the high-dose (250 microgram) test In practical terms, the low-dose test may be better at catching subtle cases, while the high-dose test remains standard in many hospitals simply because its protocol is easier to handle. The 1-microgram dose is tricky to prepare accurately from a vial designed for 250 micrograms, and small errors in dilution can affect results.
Diagnosing Primary Adrenal Insufficiency
Primary adrenal insufficiency, often called Addison’s disease, means the adrenal glands themselves are damaged, usually by autoimmune destruction. Because the glands are directly impaired, they cannot respond to ACTH at any dose, so the stimulation test tends to be quite reliable here. The meta-analysis mentioned earlier found a sensitivity of about 92% for the high-dose test in primary adrenal insufficiency.4The Journal of Clinical Endocrinology & Metabolism. ACTH Stimulation Tests for the Diagnosis of Adrenal Insufficiency: Systematic Review and Meta-Analysis The low-dose test has also shown high diagnostic sensitivity and specificity in this population, including in people with preclinical disease who do not yet have obvious symptoms.5PubMed. Low dose (1 microg) ACTH test in the evaluation of adrenal dysfunction in pre-clinical Addison’s disease
For most endocrinologists, a clearly abnormal result in a patient with suggestive symptoms and autoantibodies against the adrenal cortex is enough to confirm the diagnosis and start treatment with replacement cortisol.
The Trickier Problem of Secondary Adrenal Insufficiency
Secondary adrenal insufficiency arises when the pituitary gland does not produce enough of its own ACTH. The adrenal glands are structurally intact but have shrunk from underuse. This is far more common than primary insufficiency, because it can happen after stopping long-term steroid therapy, after pituitary surgery, or because of a pituitary tumor. The ACTH stimulation test works here on a somewhat indirect principle: if the adrenals have been deprived of ACTH stimulation for long enough, they will have atrophied and will respond poorly to an external dose. But if the pituitary problem is recent, the adrenals may not have had time to shrink, and they can still mount a normal response to injected ACTH. The test result looks fine even though the patient’s own ACTH production is inadequate.
This is the test’s well-known blind spot. The meta-analysis data confirmed that both high- and low-dose versions are adequate for ruling in secondary adrenal insufficiency when the result is clearly abnormal, but neither is reliable for ruling it out when the result looks normal.6The Journal of Clinical Endocrinology & Metabolism. ACTH Stimulation Tests for the Diagnosis of Adrenal Insufficiency: Systematic Review and Meta-Analysis A review in Biomedicines noted that the diagnostic accuracy for secondary insufficiency varies widely across published studies, partly because of differences in dose, timing, and lab assays used.7PubMed Central. ACTH Stimulation Test for the Diagnosis of Secondary Adrenal Insufficiency: Light and Shadow
When there is strong clinical suspicion but the ACTH stimulation test comes back normal, doctors sometimes turn to the insulin tolerance test, which is considered the gold standard. That test deliberately lowers blood sugar with insulin, which triggers a stress response through the entire hypothalamic-pituitary-adrenal axis. It is a more complete assessment but carries real risks, especially in people with seizure disorders or heart disease, and is unpleasant enough that many centers avoid it. After pituitary surgery, the low-dose ACTH test has shown better agreement with the insulin tolerance test than the high-dose version, making it a more practical choice in that particular setting.8PubMed. A comparison between the 1-microg adrenocorticotropin (ACTH) test, the short ACTH (250 microg) test, and the insulin tolerance test in the assessment of hypothalamo-pituitary-adrenal axis immediately after pituitary surgery
When the Lab Assay Changes the Answer
One underappreciated issue is that not all cortisol assays produce the same number. Older immunoassays tend to cross-react with cortisol-like molecules in the blood, giving higher readings. Newer, more specific assays, including tandem mass spectrometry, tend to give lower values. A study comparing the Roche Elecsys Cortisol II immunoassay with mass spectrometry derived different cutoff values: roughly 426 versus 411 nmol/L at 30 minutes, and 485 versus 470 nmol/L at 60 minutes.9PubMed Central. Determination of cortisol cut-off limits and steroid dynamics in the ACTH stimulation test: a comparative analysis using Roche Elecsys Cortisol II immunoassay and LC-MS/MS Another study found that immunoassay measurements were about 12% lower than mass spectrometry for total cortisol, with the direction of the discrepancy depending on the specific platform.10The Journal of Clinical Endocrinology & Metabolism. Free and Total Plasma Cortisol Measured by Immunoassay and Mass Spectrometry Following ACTH1–24 Stimulation in the Assessment of Pituitary Patients
The practical problem is that many hospitals still use the traditional 500-nmol/L cutoff regardless of which assay they run. As labs switch to more specific assays, using an old cutoff designed for a less specific assay will misclassify some patients as insufficient who are actually fine. Research has explicitly called for assay-specific cutoffs.11Journal of the Endocrine Society. New Cutoffs for the Biochemical Diagnosis of Adrenal Insufficiency after ACTH Stimulation using Specific Cortisol Assays If you receive an ACTH stimulation test result that is borderline, it is worth asking your doctor whether the cutoff used matches the assay your lab actually runs.
Factors That Can Throw Off Results
Cortisol circulates in the blood mostly bound to a protein called cortisol-binding globulin (CBG). Standard lab tests measure total cortisol, bound and unbound together. Any condition that raises or lowers CBG will shift the total number without changing how much cortisol is actually available to tissues. Estrogen-containing oral contraceptives raise CBG substantially, which inflates total cortisol readings and can make the test appear normal when adrenal function is actually borderline. Liver cirrhosis lowers CBG, pushing total cortisol readings down and potentially triggering a false diagnosis of adrenal insufficiency.12PubMed. Long-acting porcine ACTH stimulated salivary cortisol reduces the overdiagnosis of adrenal insufficiency compared to serum cortisol in cirrhosis liver
Medications can also interfere. Exogenous glucocorticoids, including inhaled steroids, topical creams, and joint injections, suppress the body’s own ACTH production and can lead to a blunted test result that reflects drug-induced suppression rather than intrinsic adrenal disease. A recent study emphasized that careful preparation, including stopping confounding medications before measuring morning cortisol, can often sort patients well enough that many do not need the stimulation test at all.13PubMed Central. ACTH Stimulation Testing Is Often Unnecessary After Appropriate Patient Preparation Prior to Screening Cortisol Measurement The timing of the test matters, too. Cortisol has a strong daily rhythm, peaking early in the morning. Most protocols call for testing before 9 or 10 a.m. to take advantage of that natural peak, which gives the most interpretable baseline.
Salivary Cortisol as an Alternative
Because total serum cortisol is so sensitive to changes in binding proteins, researchers have explored salivary cortisol as an alternative measurement during the ACTH stimulation test. Cortisol in saliva closely reflects the unbound, biologically active fraction of cortisol in the blood and is not affected by CBG levels.14The Journal of Clinical Endocrinology & Metabolism. Salivary Cortisol Response to ACTH Stimulation Is a Reliable Alternative to Serum Cortisol in Evaluating Hypoadrenalism One study showed that stimulated salivary cortisol levels closely correlated with serum total cortisol and had comparable diagnostic performance, proposing a cutoff of about 13.2 nmol/L for diagnosing adrenal insufficiency via saliva.15Endocrinology and Metabolism. Stimulated Salivary Cortisol as a Noninvasive Diagnostic Tool for Adrenal Insufficiency In patients on oral contraceptives or with liver disease, salivary cortisol performed more reliably than serum cortisol, with stimulated salivary values that did not differ between healthy controls and women on the pill, even though serum values diverged significantly between those groups.16Journal of the Endocrine Society. OR30-02 Salivary Cortisol Response To The High Dose ACTH Stimulation Test Provides A Reliable Alternative To Serum Cortisol Measurement In The Evaluation of Suspected Hypoadrenalism, And Is Unaffected By Cortisol-Binding Concentration
Salivary testing is noninvasive and does not require a blood draw, which makes it appealing for repeat testing or for patients with difficult venous access. It has not yet replaced serum cortisol in standard clinical practice, partly because labs need validated reference ranges for the specific salivary assays they use, and those ranges are still being established in many settings.
Screening for Congenital Adrenal Hyperplasia
The ACTH stimulation test is not only for diagnosing adrenal insufficiency. It also plays a central role in evaluating nonclassic congenital adrenal hyperplasia (NCCAH), a milder genetic condition in which a partially functioning enzyme leads to excess production of adrenal androgens. Rather than measuring cortisol alone, the test here tracks a precursor hormone called 17-hydroxyprogesterone (17-OHP). Injecting ACTH amplifies the metabolic bottleneck caused by the enzyme deficiency, producing a characteristic spike in 17-OHP.
A study using modern mass spectrometry assays found that a baseline 17-OHP cutoff of about 6 nmol/L could screen for NCCAH with roughly 96% sensitivity and specificity, potentially sparing many patients the need for a full stimulation test.17Journal of the Endocrine Society. Screening for Nonclassic Congenital Adrenal Hyperplasia in the Era of Liquid Chromatography-Tandem Mass Spectrometry However, relying solely on 17-OHP measurements, whether baseline or stimulated, can lead to false positives, especially when older immunoassay methods are used. A study in Clinical Endocrinology cautioned that the definitive diagnosis should be confirmed with genetic testing when results are ambiguous.18PubMed. The diagnosis of nonclassic congenital adrenal hyperplasia due to 21-hydroxylase deficiency, based on serum basal or post-ACTH stimulation 17-hydroxyprogesterone, can lead to false-positive diagnosis
The Test in Critical Illness
One of the most contested uses of the ACTH stimulation test is in the intensive care unit, where critically ill patients sometimes develop what is called critical illness-related corticosteroid insufficiency (CIRCI). The concept is that during severe sepsis or other major physiological stress, the body’s cortisol response may be inadequate relative to the severity of the illness, even if absolute cortisol levels are not obviously low.
International guidelines from the Society of Critical Care Medicine and the European Society of Intensive Care Medicine acknowledged that no single test reliably diagnoses CIRCI. They suggested that a change in cortisol of less than 9 µg/dL after 250 micrograms of cosyntropin, or a random cortisol below 10 µg/dL, could be used as rough diagnostic markers, but the task force could not reach agreement on endorsing a definitive test.19PubMed. Guidelines for the diagnosis and management of critical illness-related corticosteroid insufficiency (CIRCI) in critically ill patients (Part I): Society of Critical Care Medicine (SCCM) and European Society of Intensive Care Medicine (ESICM) 2017 The difficulty is compounded by the fact that adrenal function in ICU patients is dynamic and changes over the course of illness. Research tracking ACTH test results over time in critically ill patients found that a falling cortisol response during septic shock correlated with worse outcomes, suggesting that the adrenals’ ability to respond shifts as the disease evolves rather than being a fixed trait.20PubMed. Risk factors and outcome of changes in adrenal response to ACTH in the course of critical illness
For ICU physicians, this means the ACTH stimulation test is a piece of a puzzle, not a standalone answer. A single snapshot of adrenal function during critical illness may not reflect what is going on an hour or a day later.
Using Morning Cortisol to Avoid the Test Entirely
Not everyone with suspected adrenal problems actually needs a stimulation test. A simple morning cortisol blood draw can often sort patients into clear categories. Very low morning cortisol strongly suggests insufficiency, and very high morning cortisol effectively rules it out. The stimulation test is most useful for people in the gray zone, where a morning value is neither clearly normal nor clearly deficient.
A recent study using a second-generation cortisol assay proposed that a morning cortisol above about 141 µg/L could predict a normal low-dose ACTH test response with high sensitivity, reducing the number of stimulation tests needed by about a quarter compared with older guidelines. On the other end, a morning cortisol at or below roughly 61 µg/L predicted test failure with high specificity.21PubMed Central. Morning cortisol and central adrenal insufficiency: new thresholds from low-dose ACTH test and second-generation assay The exact thresholds vary by assay, which circles back to the same problem that affects the stimulation test itself: labs running different cortisol platforms need different cutoffs.
Pregnancy and the Adrenal Response
Pregnancy complicates adrenal testing in several ways. Estrogen levels rise dramatically, which in turn increases CBG and inflates total serum cortisol. At the same time, the adrenal glands become genuinely more active as pregnancy progresses. A study of pregnant women found that both basal cortisol and the adrenal response to ACTH stimulation increased across trimesters, and that salivary free cortisol was a more consistent, binding-globulin-independent measure of adrenal function than total serum cortisol in this population.22The Journal of Clinical Endocrinology & Metabolism. Assessment of Adrenal Reserve in Pregnancy: Defining the Normal Response to the Adrenocorticotropin Stimulation Test Standard cutoffs derived from nonpregnant adults should not be applied to pregnant patients without adjustment, but trimester-specific reference ranges are not yet widely established, making interpretation a judgment call.
The ACTH Stimulation Test in Veterinary Medicine
Dogs develop adrenal diseases that closely mirror human conditions, and the ACTH stimulation test is a cornerstone of veterinary endocrinology for both diagnosing hypoadrenocorticism (the canine equivalent of Addison’s disease) and monitoring treatment of hyperadrenocorticism (Cushing’s disease). A study comparing a low-dose protocol (5 µg/kg) to the standard 250 µg per dog found that one-hour post-stimulation cortisol levels were statistically equivalent between the two doses.23Journal of Veterinary Internal Medicine. Use of a Low-Dose ACTH Stimulation Test for Diagnosis of Hypoadrenocorticism in Dogs The lower dose costs significantly less, which matters in veterinary practice where cosyntropin pricing can be a genuine barrier to testing.
For monitoring dogs treated with trilostane for Cushing’s disease, however, the picture is murkier. Research found that the ACTH stimulation test was not able to reliably identify undertreated dogs, suggesting that clinical signs and other markers may be more useful than cortisol levels alone for guiding dose adjustments in treated animals.24Wiley Online Library. Comparison of methods to monitor dogs with hypercortisolism treated with trilostane This parallels the lesson from human critical care: the test is better at diagnosis than at nuanced, real-time monitoring of a dynamic condition.