Abstract: In horses, a circadian rhythm in plasma cortisol concentrations has been reported in some but not all studies. When a rhythm occurred, horses were accustomed to a management routine, comprising stabling, feeding and sometimes exercise, which may entrain a circadian pattern. In this work, we monitored plasma cortisol by collecting jugular blood through indwelling cannulae from four groups: 1): 10 untrained, unperturbed mares grazing excess pasture, bled hourly for 26 hr; 2) 4 mares housed in a barn for 48 hr before sampling every 15 min for 20–24 hr; 3) 5 mares placed in an outdoor yard for sampling every 30 min from 0930–2100 hr; and 4) 4 stabled racehorses in training, bled every 30 min from 0730–2000 hr and once the following morning at 0830 hr. Plasma cortisol showed a similarly-timed circadian rhythm (P<0.0001) in all Group 1 horses, with a peak at 0600–0900 hr, and a nadir at 1800–2100 hr. By contrast, cortisol concentrations did not vary with time in either Group 2 or 3. Neither daily mean nor peak cortisol values differed in Group 1 and 2 (i.e. bled for >= 20 hr); however nadir values were higher (P<0.05) in Group 2. In Group 4, cortisol declined (P=0.004) during the sampling period but had returned to initial concentrations the next morning. Values did not differ from those for Group 1, except between 1000 and 1300 hr when cortisol in Group 4 was lower (P<0.05). We conclude that a circadian cortisol rhythm exists in horses in the absence of any known cues imposed by humans. However, this rhythm can be obliterated by the minor perturbation of removing the horse from its accustomed environment. By contrast, the rhythm occurs in trained racehorses, suggesting either that they have adapted to their environment thereby allowing an endogenous rhythm to emerge, or that the rhythm is entrained by their daily routine. These observations highlight the difficulties in determining the cortisol status of a horse, since measurements will be affected by time of day, the occurrence of short-term fluctuations, and how accustomed the horse is to its environment.