Abstract: Different head-and-neck positions (HNPs) are discussed in relation to potential welfare issues. To evaluate the effect on welfare, seven Royal Dutch Sport horses were studied in five predetermined HNPs: (1) unrestrained (HNP1); (2) neck raised, bridge of nose around the vertical (HNP2); (3) neck lowered and considerably flexed, bridge of nose pointing towards the chest (HNP4); (4) neck raised and extended, bridge of nose in front of the vertical (HNP5), and (5) neck lowered and flexed, bridge of nose pointing towards the carpus (HNP7). A standardised exercise test (SET) of 34 min consisted of trot, canter and walk. Behaviour was recorded with a pre-defined ethogram and R-R intervals measured using telemetry. Cortisol concentrations were taken at the start, 5 and 30 min after the SET. Behaviour around the SET was scored separately. Conflict behaviours increased significantly during HNP2 when compared with HNP1, HNP4 and HNP7 during the SET, and there was significant negative anticipation before HNP2 and HNP7. The heart rate variability (HRV) frequency domain for HNP2 showed a significantly increased low frequency peak (LFpeak) compared with other HNPs, and there was a decrease in very low frequency (VLF%) compared with HNP1. HNP4 showed a significant increase in LF% and decrease in VLF% compared with HNP1. Saliva cortisol concentrations were significantly increased in HNP2 at 5 and 30 min after exercise. Increased conflict behaviour was mostly observed in HNP2, but there was a raised HRV suggesting a sympathetic shift in HNP2 and HNP4, and increased cortisol concentrations during HNP2 indicated a stress response.