AbstractThe objective was to examine behavioural and physiological reactions of cows in pens with slatted floors, when pretreatment was tethering in stalls. Ten cows were kept tethered (C), whereas 10 other cows were kept loose in pens with slatted flooring (S). Cow behaviour was observed prior to treatment, and after 4 and 8 weeks, and behaviour in two open-field tests was recorded. Serial plasma concentrations of ACTH and cortisol were measured on day 23 during a 7.5 h period. On d 24, cortisol response was measured in serial samples drawn 0.5 h prior to and for 5 h after intravenous ACTH administration. S-cows showed reduced lying duration and frequency and spent less time exploring and more time immobile in the first test. S-cows showed a slightly increased response to ACTH-injection, otherwise pituitary-adrenal axis reactions did not differ between treatment groups.