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Adrenocorticotropic hormone (ACTH) testing is used to evaluate horses for pituitary pars intermedia dysfunction (PPID), commonly known as equine Cushing’s disease. PPID is one of the most common endocrine disorders in older horses.
Many signs of PPID develop gradually or overlap with normal aging. Laboratory testing therefore plays an important role in diagnosis. Testing primarily involves measuring plasma ACTH, either as a resting concentration or as part of a thyrotropin-releasing hormone (TRH) stimulation test.
ACTH concentrations naturally fluctuate throughout the year, particularly during the seasonal autumn rise. Illness, medication use, and ongoing PPID treatment can also influence results.
Appropriate testing can help detect PPID earlier, guide treatment decisions, and support ongoing monitoring. This article reviews resting ACTH and TRH stimulation testing and the factors veterinarians consider when interpreting results.
Adrenocorticotropic Hormone (ACTH) Testing in Horses
Pituitary pars intermedia dysfunction is a degenerative disorder associated with aging in horses. The disease involves degeneration of dopaminergic neurons in the hypothalamus. [1] These nerve cells use dopamine to help control the pars intermedia of the pituitary gland, a major hormone-producing structure in the brain.
The pars intermedia produces several hormones and hormone fragments, including adrenocorticotropic hormone (ACTH). [2] ACTH acts on the adrenal glands and stimulates the production of cortisol. Cortisol helps regulate how the body uses energy, responds to stress, and controls immune activity.
When dopaminergic neurons degenerate, this control declines. The pars intermedia then produces ACTH and other related hormones in excess. This can cause higher ACTH levels in the horse’s blood, which veterinarians can measure with a blood test.
The most common test is a resting ACTH test, which is performed without first stimulating the pituitary gland. The veterinarian collects a single blood sample, and a laboratory measures the concentration of ACTH in the plasma. [1]
ACTH levels naturally change throughout the year and rise during late summer and autumn in North America. For this reason, veterinarians compare the result with reference values for the time of year. [1][2]
A result that is clearly above the expected range can support a diagnosis of PPID when it is consistent with the horse’s clinical signs. Some results fall into an intermediate range and are less conclusive. In these cases, the veterinarian may repeat the test at a later date or use a TRH stimulation test to further assess pituitary function. [1]
Signs that Warrant ACTH Testing
The hormonal changes associated with PPID can affect several body systems. When a horse develops signs consistent with PPID, a veterinarian may recommend ACTH testing to assess pituitary function.
Changes in the hair coat are one of the most recognizable signs of PPID. The pars intermedia produces hormones involved in seasonal hair growth and shedding. In horses with PPID, excess hormone production can disrupt normal coat changes and lead to hypertrichosis, or an abnormally long hair coat. [2]
PPID can also cause a range of other signs as hormone regulation becomes disrupted. Common clinical signs of PPID include: [1]
- Delayed shedding
- Loss of topline muscle
- Lethargy
- Decreased performance or exercise intolerance
- A rounded, potbellied appearance
- Increased urination and thirst
- Recurrent infections
- Laminitis
The presence of one or more of these signs does not confirm PPID. Veterinarians interpret ACTH test results together with the horse’s age, clinical signs, medical history, and the time of year to make a diagnosis.
When to Call the Veterinarian
Contact your veterinarian if an older horse develops new or worsening signs that may indicate PPID.
These signs often develop gradually and can also occur with other health conditions. A veterinary examination is necessary to determine whether endocrine testing is appropriate.
Veterinary evaluation is recommended for horses with:
- An abnormally long hair coat or delayed shedding
- Progressive muscle loss, lethargy, or reduced exercise tolerance
- Increased drinking and urination
- Recurrent infections or episodes of laminitis
- An ACTH test result that does not match the horse’s clinical signs
Contact your veterinarian promptly if laminitis is suspected. Sudden or marked lameness, reluctance to move, or other warning signs of laminitis require timely assessment.
Your veterinarian should evaluate the horse and determine the appropriate next steps.
What's your top priority with your horse's health?
What's your top priority with your horse's health?
Available Diagnostic Tests
Veterinarians primarily use ACTH testing to diagnose PPID. Other options include the TRH stimulation test and the overnight dexamethasone suppression test. [2]
Resting ACTH testing measures the horse’s baseline hormone concentration, while the TRH stimulation test evaluates how strongly the pituitary gland responds when stimulated. The overnight dexamethasone suppression test evaluates a different hormonal pathway and is now used less often. [1][2]
The most appropriate test depends on the horse’s clinical signs, age, season, medical history, and previous test results.
Table 1. PPID Diagnostic Tests
| Test | Measurement | When to Use | Considerations |
|---|---|---|---|
| Resting plasma ACTH |
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| TRH stimulation test |
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| Overnight dexamethasone suppression test |
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|
ACTH Testing
Measuring a horse’s resting ACTH concentration can help veterinarians assess how much ACTH the pituitary gland is producing under normal conditions.
For this test, the veterinarian collects blood into an appropriate tube and separates the plasma, the liquid portion of blood. A laboratory then measures the ACTH concentration in the plasma. [1]
ACTH results can be challenging to interpret because reference intervals vary throughout the year. This variation reflects the pituitary gland’s role in seasonal responses to day length.
During late summer and autumn, pituitary secretions increase as days shorten. [2] This seasonal change helps activate processes such as increasing hair length as temperatures get colder.
In North America, veterinarians use different resting ACTH thresholds based on the month of testing. Horses tested in winter, spring, and early summer have lower thresholds than horses tested in late summer or autumn. [1]
The Equine Endocrinology Group (EEG) is a group of equine clinicians and researchers that develops diagnostic and management recommendations for endocrine disorders in horses. This group recommends an interpretive zone for results that fall between PPID-unlikely and PPID-likely values. [1]
For horses in this zone, the veterinarian considers the severity of clinical signs when deciding on treatment or further testing. Horses with advanced clinical signs may begin treatment even when ACTH concentrations do not exceed the threshold for a likely diagnosis.
Table 2. Resting ACTH interpretation by time of year
| Time of Year | PPID Unlikely | Interpretive Zone | PPID Likely |
|---|---|---|---|
| January–June | < 15 pg/mL | 15 – 40 pg/mL | > 40 pg/mL |
| July | < 15 pg/mL | 15 – 50 pg/mL | > 50 pg/mL |
| August | < 20 pg/mL | 20 – 75 pg/mL | > 75 pg/mL |
| September–October | < 30 pg/mL | 30 – 90 pg/mL | > 90 pg/mL |
| November | < 15 pg/mL | 15 – 50 pg/mL | > 50 pg/mL |
| December | < 15 pg/mL | 15 – 40 pg/mL | > 40 pg/mL |
Some breeds and horse types can have highly variable ACTH concentrations even when healthy. Arabians, donkeys, and Welsh and Shetland ponies may have higher ACTH concentrations than expected for the season, potentially placing them in the interpretive zone or PPID-likely category. [1]
These animals require careful assessment of clinical signs, with test results interpreted in the context of the individual horse.
TRH Stimulation Test
Thyrotropin-releasing hormone is produced by the hypothalamus and acts on the pituitary gland. When administered during a TRH stimulation test, it prompts the pituitary to release ACTH.
Both healthy horses and horses with PPID show an increase in ACTH after TRH administration, but horses with PPID typically have a greater response because abnormal cells in the pars intermedia release excessive amounts of ACTH. [2]
Veterinarians use this pituitary response as a dynamic diagnostic test. The veterinarian collects a baseline blood sample and then administers a dose of TRH intravenously. Follow-up samples are collected at specified times, commonly 10 or 30 minutes after administration, to measure the horse’s ACTH response. [1]
In a horse with PPID, TRH administration causes an exaggerated or prolonged increase in ACTH compared with the response expected in a horse without PPID. Results must be compared with reference intervals appropriate for the sampling time, season, laboratory method, and testing protocol.
Veterinarians may use the TRH stimulation test in the following situations: [1]
- When a horse has few, mild, or early clinical signs
- When a horse is younger than the typical PPID patient, generally under 15 years of age
- When resting ACTH falls within the interpretive or PPID-unlikely zone despite clinical signs
As with resting ACTH, TRH stimulation results vary by season. [1] Horses can have a more pronounced response to TRH in late summer and autumn, which makes interpretation more difficult. During this period, a result below the PPID-unlikely threshold can help rule out PPID, whereas an elevated result may be less definitive because responses vary considerably among individual horses. [1]
Table 3. ACTH interpretation after TRH stimulation
| Time of Year | PPID Unlikely | Interpretive Zone | PPID Likely |
|---|---|---|---|
| 10 minutes after TRH | |||
| January–June | < 100 pg/mL | 100 – 200 pg/mL | > 200 pg/mL |
| July–December | < 100 pg/mL | Highly variable; no reference interval established | |
| 30 minutes after TRH | |||
| January–June | < 40 pg/mL | 40 – 90 pg/mL | > 90 pg/mL |
| July–December | < 40 pg/mL | Highly variable; no reference interval established | |
Horses with clinical signs whose test results fall within the interpretive or PPID-unlikely zone should be retested every three to six months. Repeat testing helps track disease progression so treatment can begin when appropriate. [1]
Overnight Dexamethasone Suppression Test
For many years, the overnight dexamethasone suppression test (ODST) was considered the gold standard for diagnosing PPID. This test evaluates whether dexamethasone, a corticosteroid similar to cortisol, suppresses the horse’s natural cortisol production.
After the veterinarian administers dexamethasone, a healthy horse’s body will reduce hypothalamic and pituitary signaling, which lowers cortisol production by the adrenal glands. The veterinarian measures cortisol the following morning to determine whether appropriate suppression occurred.
Horses with PPID may fail to suppress cortisol adequately after dexamethasone administration, supporting a diagnosis of PPID. [2]
The ODST has largely fallen out of favor for the following reasons: [2][3]
- Seasonally adjusted reference intervals are not available
- Dexamethasone may induce or worsen laminitis in horses with PPID and/or equine metabolic syndrome
- The test requires either two veterinary visits or hospitalization
- It may be less reliable for detecting early disease
Veterinarians may still use the ODST in selected cases as additional evidence supporting a PPID diagnosis.
Preparing Your Horse for Testing
Most horses do not require specific preparation for PPID testing. Resting ACTH measurement is a simple blood test that can be performed at any time of day. [1] However, avoid stress, excitement, or trailering immediately before testing, as these factors can increase ACTH concentrations above the horse’s normal baseline. [1]
For the TRH stimulation test, avoid feeding grain for at least 12 hours before testing. [1] TRH administration may cause the horse to cough, yawn, or show a flehmen response. These effects are typically mild and resolve as the TRH is processed by the horse’s body. [1]
Depending on the horse’s clinical signs, the veterinarian may also recommend insulin testing for equine metabolic syndrome. Some insulin-testing protocols may require overnight fasting. Follow the veterinarian’s preparation instructions for the specific tests being performed.
Medications can also influence ACTH concentrations. Tell the veterinarian about all medications and supplements the horse receives. In particular, pergolide, the medication used to treat PPID, can affect ACTH results.
Frequently Asked Questions
Here are some frequently asked questions about PPID testing in horses:
Pituitary pars intermedia dysfunction (PPID), commonly called equine Cushing's disease, is a progressive endocrine disorder associated with aging in horses. It develops when dopaminergic neurons in the hypothalamus degenerate and no longer regulate the pars intermedia of the pituitary gland properly. The resulting increase in pituitary secretions, including ACTH, contributes to the characteristic clinical signs of PPID.
Clinical signs of PPID in horses include a long or abnormal hair coat, delayed shedding, loss of topline muscle, lethargy, reduced performance, and a rounded or potbellied appearance. Affected horses may also drink and urinate more, sweat excessively, or develop recurrent infections. Because these changes can appear gradually and overlap with normal aging, laboratory testing is important when PPID is suspected.
PPID is diagnosed by considering the horse's clinical signs together with the results of endocrine testing. Measuring resting plasma ACTH is the primary diagnostic approach, while a TRH stimulation test may provide additional information in horses with early signs or inconclusive results. The horse's age, breed, medications, health status, and the time of year must also be considered when interpreting test results.
The blood test most commonly used to diagnose PPID measures the horse's resting plasma adrenocorticotropic hormone, or ACTH, concentration. The veterinarian collects blood, separates the plasma, and submits it for ACTH analysis. Horses with PPID often have elevated ACTH because the abnormal pituitary gland produces and secretes hormones without appropriate regulation.
A normal ACTH level in horses depends on the time of year the blood sample is collected. From January through June, an ACTH concentration below 15 pg/mL is considered consistent with PPID being unlikely, while the acceptable seasonal threshold increases during late summer and autumn. Results should be compared with month-specific reference intervals and interpreted alongside the horse's clinical signs.
ACTH levels in horses change throughout the year because the pituitary gland responds to season and day length. Pituitary secretions naturally rise during late summer and autumn as days become shorter, supporting seasonal changes such as winter coat growth. This seasonal autumn rise can produce higher ACTH concentrations even in horses that do not have PPID.
A horse can be tested for PPID at any time of year, provided seasonally adjusted reference intervals are used. Resting ACTH concentrations are naturally more variable during late summer and autumn, so results from this period require especially careful interpretation. The best testing time may also depend on the horse's clinical signs and whether the veterinarian is performing a resting ACTH or TRH stimulation test.
The interpretive zone on an ACTH test is the range between results classified as PPID unlikely and PPID likely. A result in this zone is not a definitive positive or negative diagnosis. Veterinarians consider the severity of the horse's clinical signs when deciding whether to begin treatment, perform additional testing, or monitor and retest the horse.
A horse can have PPID even when its resting ACTH result falls within the PPID unlikely range. This is most likely in horses with early or mild disease, when abnormal pituitary function may be more difficult to detect with a single baseline sample. If clinical signs remain concerning, the veterinarian may recommend a TRH stimulation test or repeat testing in three to six months.
A horse without PPID can have an ACTH result that appears higher than expected because of season, stress, disease, medication, or breed-related variation. Arabian horses, donkeys, Welsh ponies, and Shetland ponies may have higher ACTH concentrations than expected for the season even when healthy. These results should be assessed alongside clinical signs rather than treated as proof of PPID on their own.
A TRH stimulation test evaluates how the horse's pituitary gland responds to thyrotropin-releasing hormone. The veterinarian collects a baseline blood sample, administers TRH into the bloodstream, and takes additional samples at 10 and 30 minutes to measure ACTH. An exaggerated or abnormal ACTH response can provide additional evidence of PPID.
A TRH stimulation test may be recommended for a horse with few, mild, or early clinical signs of PPID. It can also be useful in horses younger than the typical PPID patient, including those under 15 years old. Veterinarians may select this test when resting ACTH falls within the interpretive or PPID unlikely range but the horse continues to show suspicious clinical signs.
A TRH stimulation test is not universally better than a resting ACTH test because the appropriate choice depends on the individual case. Resting ACTH requires only one blood sample and is the primary test used for PPID, while TRH stimulation can provide more information when baseline results are inconclusive. Clinical signs, age, season, and the availability of appropriate reference intervals help determine which test is most useful.
A TRH stimulation test can be performed during autumn, but positive or elevated results may be difficult to interpret. Horses naturally have a more exaggerated and highly variable response to TRH from July through December, and definitive positive reference intervals have not been established for this period. During these months, the test is generally more useful for identifying horses unlikely to have PPID than for confirming the disease.
Side effects from a TRH stimulation test may include coughing, yawning, or performing a flehmen response after TRH is administered. These reactions are generally mild and resolve as the horse processes the hormone. Horse owners should discuss the procedure and any individual health concerns with the veterinarian before testing.
The overnight dexamethasone suppression test evaluates whether a horse appropriately reduces its natural steroid production after receiving dexamethasone. A veterinarian administers dexamethasone and measures steroid levels the following morning. Horses that continue to have high cortisol levels despite receiving dexamethasone may be diagnosed with PPID.
The dexamethasone suppression test is no longer commonly used for PPID because seasonally adjusted reference intervals are unavailable and the test may be less reliable for detecting early disease. Dexamethasone may also induce or worsen laminitis in horses affected by PPID or equine metabolic syndrome. The procedure requires two veterinary visits or hospitalization, making it less convenient than resting ACTH testing.
A horse does not usually need to fast before a resting ACTH test. The test can generally be performed at any time of day without special dietary preparation. Fasting may still be required if the veterinarian plans to perform insulin or other metabolic testing at the same appointment.
A horse should not be fed grain for at least 12 hours before a TRH stimulation test. Other preparation requirements may depend on any additional endocrine tests being performed at the same time. Owners should follow the veterinarian's specific instructions rather than changing the horse's entire feeding program without guidance.
Stress can affect ACTH test results in horses by increasing the concentration above the horse's normal baseline. Stress, excitement, and transportation should be minimized before the blood sample is collected. Any significant illness or stressful event should also be considered when the veterinarian interprets the result.
Medications can affect PPID testing in horses by changing ACTH concentrations or complicating interpretation. Pergolide is especially important because it is used to treat PPID and can influence the test result. The veterinarian should be given a complete list of the horse's medications before testing.
PPID testing should be repeated when the result is inconclusive or does not match the horse's clinical signs. Horses that test within the interpretive or PPID unlikely range but continue to show signs of PPID may be retested every three to six months. Repeat testing or a TRH stimulation test can help identify disease progression and support timely treatment decisions.
PPID should not generally be diagnosed from clinical signs alone because many of its signs overlap with normal aging and other health conditions. A long hair coat, muscle loss, lethargy, and increased drinking may raise suspicion, but endocrine testing provides important diagnostic support. In horses with advanced signs and an ACTH result in the interpretive zone, the severity of those signs may influence the decision to begin treatment.
A horse can be tested for PPID and equine metabolic syndrome during the same veterinary visit. A veterinarian may recommend insulin testing when the horse's clinical signs suggest abnormal insulin regulation in addition to PPID. Because insulin testing may require overnight fasting, owners should confirm the preparation requirements before the appointment.
How often a horse should be tested for PPID depends on its clinical signs, previous results, and treatment status. Horses with suspicious signs but inconclusive results may need repeat testing every three to six months. Horses receiving treatment may also require follow-up testing so the veterinarian can assess hormone concentrations to monitor treatment success and guide any changes to medications.
Summary
PPID testing is an important part of diagnosing and monitoring pituitary pars intermedia dysfunction in horses. Resting ACTH and TRH stimulation testing are the primary diagnostic tools, but results must be interpreted in context, including the horse's clinical signs, the season, medications, and laboratory-specific reference intervals.
- Plasma ACTH is the most commonly used blood test for PPID in horses
- ACTH concentrations naturally increase during the seasonal autumn rise, which can complicate interpretation
- TRH stimulation testing can provide additional information when resting ACTH results are inconclusive
- Early PPID can be difficult to diagnose, and a normal resting ACTH result does not always rule out disease
- Abnormal hair coat, muscle loss, increased drinking and urination, and recurrent laminitis can increase suspicion for PPID
- Repeat or follow-up testing may be appropriate when results are borderline or do not match the horse's clinical signs
References
- Hart. K. et al. Recommendations for the Diagnosis and Management of Pituitary Pars Intermedia Dysfunction (PPID). Equine Endocrinology Group. 2023.
- Spelta. C. Equine Pituitary Pars Intermedia Dysfunction: Current Perspectives on Diagnosis and Management. Veterinary Medicine: Research and Reports. 2015.
- Rendle. D. Update on the Diagnosis and Management of Pituitary Pars Intermedia Dysfunction in Horses. Livestock. 2013.
