Because testing for hormonal disease in horses is often triggered by the horse developing laminitis, the effect of pain on test results is always a concern.
A 2020 German study looked at hospitalized horses in pain from various conditions including colic, laminitis and orthopedic conditions. All horses had been in the clinic for at least 24 hours. Horses were 15 years old or younger with no clinical signs of PPID (Cushing’s disease).
They were tested by both baseline ACTH and ACTH after TRH stimulation, both while in pain and after they had recovered. There was no difference in test results regardless of pain intensity, so no effect of pain in this study.
However, timing may be important. This study found acute pain resulted in markedly elevated cortisol and insulin resistance. Since acute stress, wounds etc. can cause the adrenal glands to release cortisol without ACTH increase, it’s unclear from that study if acute pain will influence ACTH – although it certainly increases insulin.
A 1980 study on the responses to a low grade electrical current showed there was an ACTH response to this type of acute pain. We also know that both ACTH and cortisol levels are more likely to be greater than normal ranges in acute illness.
In summary, while there are still some gaps in the available information, testing for both ACTH and insulin levels should be avoided in the first 24 hours after onset of a painful condition. Beyond that time, ACTH can probably be tested without interference. Insulin may be tested after the pain has stabilized, even if not gone entirely.
Eleanor Kellon, VMD
Dr. K's Horse Sense 
Good question! As far as I know, no one has looked at this. In fact, the exact mechanism behind how phenylbutazone lowers thyroid hormones is unknown although we do know the thyroid gland remains responsive to TSH. In fact, one study found it was over responsive https://pubmed.ncbi.nlm.nih.gov/6682298/ . All drugs in this category have the same effect. Given the lack of details, I would wait to do the TSH stimulation test. If you need alternate pain relief, try Uckele Phyto-Quench pellets (not powder).
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As far as we know, the only thing affected by phenylbutazone is thyroid testing, and that lasts about two weeks.
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So. Would I need two clear weeks off Bute before a TRH test for ACTH levels?
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If the horse was on a painkiller like bute at the time of testing would this affect the results? I can’t work this out. It wouldn’t affect the levels of cortisol or ACTH caused by the injury\illness. Or would it?
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