Animals respond to a moving floor during transport by stepping in different directions to maintain their balance, but little is known about the importance of different types of movement. Four sheep were restrained in a crate on a platform that could be programmed to provide the various movements that simulated the motion of a ship. They were exposed to three movement types, Pitch, Roll, and Heave, or a Control treatment, in pairs for 30min periods in a changeover design. The orientation and frequency of stepping movements was recorded from videos made during the treatments and heart rate responses were monitored. Heave produced the biggest stepping responses, in the forelimb. Sheep stepped most commonly straight forwards and backwards with the fore limbs, then forwards, backwards and sideways with the hind limbs. When they did make lateral stepping movements, they moved their feet more outwards than inwards, presumably as this maintained balance more effectively. Stepping movements were associated with reduced high frequency heart beats, suggesting an associated negative emotion. Sheep on the left side of the crate showed some evidence of greater stress than those on the right, regarding their limb movement preferences and heart rate variability responses. This may be because animals on the left side of the crate had no other sheep in their left eye vision, signals from which are processed by the right brain hemisphere and are associated with stress responses. In conclusion, when subjected to simulated ship movement, sheep produced stepping and heart rate responses that were connected and indicated negative emotions.