Reasons for performing study: Previous reports suggest that adrenocorticotrophic hormone (ACTH) degrades rapidly, limiting its use as a diagnostic test. Objectives: This study quantified effects of processing delays on ACTH concentrations and investigated the addition of N-phenylmaleimide (maleimide), a protease inhibitor, as a means of reducing ACTH degradation. Study design: Experimental study. Methods: Venous blood was collected from 8 healthy horses and 8 horses with pituitary pars intermedia dysfunction (PPID) with a range of ACTH concentrations. Baseline ACTH concentrations were established immediately using a chemiluminescent assay. Plasma samples were then: 1) centrifuged immediately, 2) centrifuged immediately with the addition of maleimide, or 3) allowed to separate by gravity followed by the addition of maleimide, before all samples were stored at 22Â°C and analysed at 4, 8, 24 and 48h post collection. A linear mixed effects model and Bland-Altman analyses were performed. Significance was set at P < 0.05. Results: No significant effect of plasma treatment (P = 0.1) on change in ACTH concentration was identified. However, significant effects of horse health status (P<0.001) and time (P<0.001) on change in ACTH concentration were identified. No significant interactions were found. Significant decreases in ACTH concentration occurred in horses with PPID between 4 and 8h after blood collection. In non-PPID horses, the decrease in ACTH concentration over time was not significant. Agreement with baseline values decreased over time and was greater for non-PPID horses than for PPID horses. Conclusions: Clinically useful results are still obtained if ACTH concentration is measured up to 48h after sample collection. Allowing samples to separate by gravity rather than centrifugation did not have a significant effect on ACTH concentration, and the addition of maleimide was of no benefit.