The results of few studies on tryptophan supplementation conducted in horses are contradictory and none of these studies have shown that tryptophan is an effective calmative in horses. The objective of the present study was to determine changes in hematological parameters, plasma lactate, serum cortisol and biochemical profile, as well as the behavior of untreated and calmative treated horses under stressful conditions in order to determine the effects of the applied calmative. The study also aims to confirm the importance of a 24 hour rest after work. The same six horses were on duty twice, Trial I and Trial II, under stressful conditions. Blood samples were collected three times, at pretransport, post-stress and resting phase. Horses' behavior was observed carefully by the riders. The study showed that most changes of the hematological and biochemical profiles reflect the response to exercise as horses were physically active while being on duty. The effect of the calmative is mainly reflected by a minor extent of changes of measured parameters, as determined by comparison at individual sampling phases. According to the observations of horses' behavior, we may conclude that the use of calmatives did calm the horses. The study confirmed that horses need at least 24 hours of rest after working