Adrenocorticotropic hormone (ACTH) challenges are frequently performed in repetition when evaluating stress or welfare in animals. To our knowledge, the repeatability of ACTH challenges in cattle, though fundamental to further studies of this type, has not yet been the subject of research. Therefore, the objective of this study was to evaluate the repeatability of ACTH challenges in fattening bulls of different horn status. 81 bulls were subjected to three consecutive ACTH challenges. The first challenge (C1) was performed in calves aged 1.5 months. Subsequently, animals were characterized as high or low cortisol responders and either disbudded or left with horns. They were then assigned to one of three rearing groups: a horned group (H+), a disbudded group (H-), and a mixed group (M; 50% horned and 50% disbudded), with each group containing an equal number of high and low responders. The second ACTH challenge (C2) was performed at the age of 11 months. Time of day (TOD) of challenge was either fixed (ST = same TOD) or alternated (AT = alternate TOD) between C1 and C2. The third ACTH challenge (C3) was performed 7 days after and at the same TOD as C2. Saliva samples were taken 60 and 30 minutes before and 30, 60, 90, 120, and 150 minutes after each intravenous ACTH injection. The area under the curve (AUC) was calculated with respect both to ground (AUCG) and to increase (AUCI). The AUCI increased markedly between C1 and C2 (P < 0.05) in ST bulls, and no effects were observed for AUCG between C1 and C2 in ST or AT bulls, nor for any AUC between C2 and C3 (P > 0.1). The overall repeatability of AUCG and AUCI between C1 and C2, reflecting the repeatability between two different physiological states, was poor and moderate, respectively, for ST bulls (AUCG: r = 0.24, P > 0.1, Intraclass correlation coefficient (ICC) = 0.21; AUCI: r = 0.48, P < 0.01, ICC = 0.41) and lacked in AT bulls (AUCG: r = 0.07, P > 0.1; ICC = 0.03; AUCI: r = 0.08, P > 0.1, ICC = 0.06). The repeatability of AUCG and AUCI between C2 and C3, reflecting the repeatability within the same physiological state, was moderate (AUCG: r = 0.59, P < 0.001; ICC = 0.53; AUCI: r = 0.58, P < 0.001, ICC = 0.52). Assignment to high and low responder groups based on peak cortisol concentration in C1 did not persist over time. H+ bulls showed higher AUCI in C2 and C3 (P < 0.1 and P < 0.05, respectively) than H- bulls. The M group differed from the H- group only in C3 (P < 0.05). Thus, the effect of horn status on ACTH challenges needs further investigation. In conclusion, our results report poor repeatability of the cortisol response to ACTH challenges for challenges performed in different physiological states and moderate repeatability for challenges performed within the same physiological state. Moreover, they point out the importance of standardization of TOD when performing repeated ACTH challenge.