Background: Determination of plasma adrenocotrophic hormone (ACTH) concentration (endogenous or thyrotropin-releasing hormone [TRH] stimulation test) is the most commonly used diagnostic test for pituitary pars intermedia dysfunction (PPID) in horses. Because ACTH is unstable, samples often are frozen to be shipped to laboratories or to allow for batch analysis of research samples. However, the effect of multiple freeze-thaw cycles on equine ACTH is unknown.Objective: To determine the effects of multiple freeze-thaw cycles on immunoreactive ACTH concentration.Animals: Twenty-eight horses ranging from 10 to 27 years of age were used.Methods: Prospective study. Horses were divided into 4 groups: group 1, PPID-negative, without TRH stimulation; group 2, PPID-negative, with TRH stimulation; group 3, PPID-positive, without TRH stimulation; and group 4, PPID-positive, with TRH stimulation. Whole blood was collected from each horse at baseline or 30 minutes after TRH stimulation. Immunoreactive plasma ACTH concentration was determined using a chemiluminescence assay. Plasma samples then were frozen at −80 C >24 hours, thawed at 4 C and reanalyzed for 5 freeze-thaw cycles. Changes in plasma ACTH concentration were analyzed using a linear mixed-effect model.Results: Significant effects of freeze-thaw cycles (P = .001) and PPID status (P = .04) on plasma ACTH concentration were observed, but no significant effect of TRH stimulation was identified.Conclusions and Clinical Importance: The plasma ACTH concentration is altered by freeze-thaw cycles, and the effect is observed sooner in horses with PPID. To