Adrenal Hyperfunction - Cushing Syndrome

Last Literature Review: May 2021 Last Update:

Medical Experts


Cushing syndrome (CS) results from chronic exposure to excess cortisol, most commonly from the use of exogenous corticosteroids such as prednisone and prednisolone.  Endogenous CS is uncommon and is most often caused by pituitary adenomas that secrete adrenocorticotropic hormone (ACTH), which results in adrenal hyperfunction and an overproduction of cortisol (illness with this etiology is called Cushing disease).   Less frequently, CS is caused by hormone-releasing extrapituitary tumors or primary adrenal disease.  CS is associated with increased morbidity and mortality, which can be reduced with early diagnosis and management.   The clinical presentation of CS is broad, and although diagnosis is often straightforward in advanced cases, mild cases may be challenging to diagnose.  Diagnosis is based on biochemical evaluation of cortisol concentrations in cases of clinical suspicion for CS. 

Quick Answers for Clinicians

What are the clinical features of Cushing syndrome?

Many signs, symptoms, and other conditions are associated with Cushing syndrome (CS).  Although not entirely specific, the following features are most useful in discriminating CS from other conditions :

  • Easy bruising
  • Proximal myopathy
  • Facial plethora
  • Reddish purple striae (>1 cm wide)
  • Weight gain with decrease in height percentile (in children)

However, most of the preceding features are not highly sensitive for CS. 

Many other common symptoms of CS have low specificity because they overlap with those seen in other diseases or are highly prevalent in the general population.  Additional clinical features of CS include a dorsocervical fat pad, supraclavicular fullness, facial fullness, depression, irritability, impaired memory, fatigue, obesity, hirsutism, menstrual disorders, and poor skin healing.  More common diseases with clinical symptoms that may overlap with those of CS include hypertension, osteoporosis, diabetes mellitus, and polycystic ovary syndrome. 

Which drugs or other substances can interfere with testing for Cushing syndrome?

The substances that interfere in the assessment of Cushing syndrome (CS) vary depending on the sample type and methodology of the test being used. The following list of potential interferences, although not comprehensive, should be considered when evaluating an individual for CS.

Testing Possible Result Drugs Mechanism


(If serum cortisol is assessed during DST, then drugs affecting serum cortisol testing should also be considered)

False positive









Induction of CYP3A4 accelerates dexamethasone metabolism
False negative







Inhibition of CYP3A4 impairs dexamethasone metabolism
Serum cortisol False positive



Increases cortisol-binding protein
Urinary free cortisol False positive



Interferes with chromatographic methods (eg, HPLC and LC-MS/MS)
Some synthetic glucocorticoids Cross-reacts in immunoassays



Chewing tobacco

Inhibits 11β-HSD2
Salivary cortisol False positive



Chewing tobacco

Inhibits 11β-HSD2

11β-HSD2, 11β-hydroxysteroid dehydrogenase type 2; DST, dexamethasone suppression testing; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry

Source: ES, 2008 

How do cortisol concentrations change over the course of the day, and how does this affect laboratory testing?

Cortisol concentrations generally rise and fall based on circadian rhythm; concentrations reach a nadir around midnight and peak soon after awakening (generally between 0600 hours and 0800 hours).  Because of this natural fluctuation, the sample collection for cortisol assessment must be timed correctly for either the peak (eg, collection in the morning after a dexamethasone suppression test [DST]) or the nadir (eg, late-night salivary cortisol collection [LNSC]).  Alternately, a measure of cortisol excretion throughout an entire day (eg, 24-hour urine assay) can be used. 

Cortisol concentrations rise with stress, which also affects sample collection.  For instance, conditions that raise psychological or physical stress (eg, pain, anxiety, hypotension, infection) can result in artificially elevated cortisol concentrations. 

When is genetic testing appropriate in Cushing syndrome?

Familial forms of Cushing syndrome (CS) may present either in isolation or in association with certain familial syndromes such as multiple endocrine neoplasia 1 (MEN1) syndrome.  Genetic testing may be necessary if a patient with CS has multiple family members with endocrine tumors, as often occurs with MEN1 syndrome. 

In addition to these associations with germline variants, CS often has associations with somatic variants, including gain-of-function variants in the USP8 gene and activating variants in the PRKACA gene.  The latter may cause primary pigmented nodular adrenocortical disease (PPNAD), which may lead to cyclic CS in pediatric patients.  A variant in the GNAS gene may lead to infantile CS. 

How do clinical presentation and diagnosis of Cushing syndrome differ in pediatric patients?

The most common symptom in children is weight gain with a lack of associated height gain ; evaluation for CS is not indicated in children with obesity unless their growth rate has also slowed. 

In children older than 6 years, CS is likely caused by a pituitary tumor that produces excess adrenocorticotropic hormone (ACTH).  In children younger than 6 years, an adrenal etiology is more common. 

When performing an initial evaluation for CS, the adult cutoffs may generally be used for urinary free cortisol (UFC) because most pediatric patients being assessed for CS are >45 kg (ie, these patients have weights comparable to those of adults).  Dexamethasone suppression testing (DST) also follows the adult protocol, although the dose of dexamethasone may be adjusted for patients weighing <40 kg. If a midnight serum cortisol test is performed by collection from an indwelling intravenous (IV) catheter, the cutoff is >4.4 µg/dL in children, as opposed to the >7.5 µg/dL cutoff used in adults. 

Indications for Testing

Testing for CS should be considered in the following populations:

  • Patients with multiple features of CS, particularly when the features are progressive and more predictive of CS 
  • Patients with adrenal incidentalomas  
  • Patients with features associated with CS that are unusual for their age (eg, hypertension, osteoporosis) 
  • Children with increasing weight and decreasing height percentiles 

Laboratory Testing

Diagnostic Testing

Because exogenous corticosteroid use can cause features of CS, a thorough drug history should be obtained before pursuing biochemical testing for CS.  Normal cortisol ranges vary significantly depending on the assay methodology, and different assays may have different confounding factors (eg, cross-reactivity of metabolites or potential drug interference).  Thus, results must be interpreted within the context of the normal ranges for the specific assays used. 

Initial Biochemical Testing

The tests recommended to initially evaluate a patient’s cortisol concentrations are urinary free cortisol (UFC), late-night salivary cortisol (LNSC), and dexamethasone suppression testing (1 mg overnight dexamethasone suppression testing [ODST] or 48-hour 2 mg/d low-dose DST [LDDST]).  These tests have similar diagnostic accuracy and should be selected based on suitability for the patient. 

Biochemical Tests for Cushing Syndrome
Initial TestUrinary Free CortisolLate-Night Salivary CortisolDSTa
Test protocolPatient provides 2 24-hr urine collectionsPatient provides saliva sample between 2300 and 0000 hrs on 2 different occasions

1 mg ODST

1 mg of dexamethasone is given between 2300 and 0000 hrs; serum cortisol is measured between 0800 and 0900 hrs

48-hr, 2 mg/d LDDST

0.5 mg of dexamethasone is given at 6-hr intervals over 48 hrs; serum cortisol is measured 2-6 hrs after the last dose

Circumstances that may lead to false-positive results

High fluid intake (≥5 L)

Conditions that increase cortisol production (eg, depression, alcohol use disorder, morbid obesity, DM)

Use of certain substances (eg, carbamazepine, fenofibrate, certain glucocorticoids, chewing tobacco, licorice)

Not fully characterized but may result from use of chewing tobacco, use of licorice, smoking, or stress

Alcohol use within the past 2 wks

Use of oral estrogen within the past 6 wks

Use of medications that increase dexamethasone metabolisma

Circumstances that may lead to false-negative results

Severe renal impairment

Cyclic disease

Mild hypercortisolism (consider salivary cortisol test)

Not fully characterized

Critical illness

Nephrotic syndrome

Use of medications that decrease dexamethasone metabolisma


Recommended test for pregnant women

Not recommended for patients with adrenal incidentaloma and clinical suspicion for mild CS

Results are not affected by medications that increase CBG (eg, oral estrogen, mitotane)

Same cutoff may be used for pediatric and adult patients

Not fully characterized

May be unreliable in patients with disrupted circadian rhythms (eg, shift workers, patients with depression, patients with critical illness)

1 mg ODST is suggested test for patients with renal failureLDDST is more accurate in individuals with other conditions that may raise cortisol (eg, depression, alcohol use disorder, morbid obesity, DM)

DST is not recommended for:

  • Patients taking antiepileptic drugs
  • Patients suspected of having cyclic CS

For pediatric patients weighing >40 kg, adult protocol and cutoff can be used

aDST may be useful to confirm administration or to help interpret results of cortisol testing in patients taking medications that affect dexamethasone metabolism.

CBG, cortisol-binding hormone; DM, diabetes mellitus

Source: Nieman, 2008 ; Pappachan, 2017 ; Nieman, 2018 

If the results of initial testing are normal in a patient with a high pretest probability of CS, then further evaluation by an endocrinologist is recommended.  If a patient with normal results did not have a high pretest probability of CS, then reevaluation is suggested in 6 months if symptoms have progressed.  If results are abnormal, additional testing should be considered based on the patient’s clinical presentation. 

Confirmatory Testing

Exclusion of Other Conditions

When initial testing suggests CS, other etiologies of hypercortisolism should be excluded. If exogenous glucocorticoid use has not yet been ruled out, patient history should be thoroughly assessed.  Further testing should be performed to rule out pseudo-Cushing syndrome (PCS).   

Individuals with PCS may have both clinical features and initial biochemical evaluation results that are suggestive of CS.   However, these individuals do not have endogenous pathologic hypercortisolism.  Causes of PCS may include morbid obesity, extreme exercise, stress, hypertension, pregnancy (in the second or third trimester), alcohol use disorder, uncontrolled DM, polycystic ovary syndrome (PCOS), eating disorders, and certain psychiatric disorders such as depression.   If a patient has one of these conditions, treating the condition and reassessing cortisol concentrations may help identify PCS.  

There is currently no gold standard for differentiating between CS and PCS.  A thorough clinical evaluation and the selection of an optimal screening test, based on a detailed understanding of the performance of each test in different clinical settings, is necessary for diagnostic accuracy. 

Biochemical Confirmation

In patients with abnormal results in the initial evaluation, a second biochemical test for CS should be performed using a different technique than that used for the initial test (see Biochemical Tests for Cushing Syndrome table).  If the second test also yields abnormal results and the patient had a high pretest probability of CS, a diagnosis of CS is likely.  Because of the complexity of test interpretation, determination of cause, and subsequent treatment considerations, referral to endocrinologists with expertise in CS is highly recommended.

In certain situations, a variation of the LDDST or a midnight serum cortisol test can be added to help confirm a diagnosis of CS. The variation of the LDDST entails administration of cortisol-releasing hormone (CRH) after the last dose of dexamethasone and before measuring cortisol. This test can be useful in patients who have equivocal UFC results.   The midnight serum cortisol test (either sleeping or awake) is difficult to perform and thus is not recommended as an initial test.  However, it may be useful in two specific scenarios: in patients with a high clinical index of suspicion for CS but with normal UFC and DST results and in patients with a low clinical index of suspicion but with abnormal DST results and slightly increased UFC.  In the former situation, a sleeping level of >1.8 µg/dL or an awake level of >7.8 µg/dL indicates a higher possibility of CS; in the latter, a sleeping level of <1.8 µg/dL or an awake level of <7.8 µg/dL essentially rules out CS. 

Determination of Etiology

After a diagnosis of CS, follow-up testing should be performed to determine the underlying cause of excess cortisol production. 

Biochemical Testing

Measuring the ACTH concentration can help to determine if a patient has ACTH-independent or ACTH-dependent CS. 

Clinical Significance of ACTH Results
Serum ACTH ConcentrationsProbable ClassificationProbable Disease EtiologyAdditional Follow-Up Testing
<10 pg/mL (<2.2 pmol/L)ACTH-independent CSAdrenal pathology, usually unilateral, although bilateral gland involvement is possiblen/a
10-20 pg/mL (2.2-4.4 pmol/L)UncertainUncertain

CRH stimulation testing may help differentiate between ACTH-independent and ACTH-dependent disease

Disease of pituitary origin will respond to CRH stimulation, whereas disease of adrenal origin will not

>20 pg/mL (>4.4 pmol/L)ACTH-dependent CSPituitary adenoman/a
>500 pg/mL (>110 pmol/L)Ectopic ACTH-driven CSNeuroendocrine tumors

Consider testing for increased neuroendocrine hormones (eg, calcitonin, gastrin, chromogranin A) to support diagnosis of ectopic ACTH-driven CS

Consider performing HDDST to help differentiate between ectopic ACTH-driven CS and CS of pituitary origin

HDDST, high-dose dexamethasone suppression testing; n/a, not applicable

Source: Pappachan, 2017 


When the underlying cause of disease is uncertain, imaging studies can be performed on the pituitary gland for ACTH-dependent disease to help differentiate CS of pituitary origin from ectopic ACTH-driven CS.  If the biochemical and imaging results are still inconclusive, bilateral inferior petrosal sinus sampling (BIPSS) should be performed to confirm the diagnosis and inform a management approach. 

Imaging studies on the adrenal glands may aid in determining the pathology of ACTH-independent disease, such as adrenal adenoma or cancer, which can guide treatment decisions.    Adrenal venous sampling (AVS) may also help to determine which adrenal gland is causing the cortisol overproduction before surgical or medical management. 

ARUP Laboratory Tests

Diagnostic Testing
Determination of Etiology


Additional Resources