Abstract Background It is unknown whether short‐term transportation affects endocrine responses similarly in horses with and without insulin dysregulation (ID). Objectives To characterise the effect of short‐term transportation on stress parameters and insulin responses to an oral sugar test (OST) in horses with and without ID. Study design Longitudinal cohort study. Methods Fourteen adult non‐pregnant, non‐PPID mares of mixed light breeds were grouped as either ID ( n = 7) or non‐ID ( n = 7) based on endocrine testing. Over 2 weeks, horses were transported once, in groups of 3–4 in a horse trailer on a round‐trip journey of ~1.5 h. Blood and saliva were collected 24 h and 1 h pre‐transportation, directly after unloading and 15 min, 1 h, 3 h plus 24 h post‐transportation. An OST was performed 24 h pre‐transportation and 3 h post‐transportation with a pre‐ (T0) and post‐OST sample collected 60 min later (T60). Heart rates and rectal temperatures were also collected throughout the study. Serum insulin, serum cortisol, and plasma glucose were measured using validated assays. Repeated measures ANOVA were used to determine differences after transportation and between ID and non‐ID horses. Non‐normal data were log‐transformed and multiple comparisons were adjusted using Bonferroni post hoc tests. Results Mean insulin was higher in ID horses versus non‐ID horses (mean = 109.9 μU/mL vs. 30.2 μU/mL, p < 0.001; 95% CI for mean difference = [55.6–107.7 μU/mL]). Mean serum insulin increased following OST at T60 in ID horses pre‐ (154.6 μU/mL, p = 0.04; 95% CI = [86.3–223.0 μU/mL]) and post‐transportation (284.6 μU/mL, p = 0.03; 95% CI = [114.3–454.8 μU/mL]). Non‐ID horses had a mean OST T60 insulin post‐transportation of 56.6 μU/mL (95% CI = [29.1–84.1 μU/mL]); above recognised threshold [45 μU/mL] for ID diagnosis. Main limitations Small number of horses, only mares used, and OST not performed immediately post‐transportation. Conclusions Performing an OST 3 h following short‐term transportation may result in inaccurate ID status.