An experiment was conducted examining heart rate and behavioural responses of previously unhandled horses to novel visual, auditory and olfactory stimuli. The horses were taught to expect a food reward from a container in a test arena, in addition to either a two minute visual test (using a traffic cone), a two minute auditory test (using white noise), or a two minute olfactory test (conducted by applying eucalyptus oil to the inside of the food container). Less time was spent eating during each test, when compared to the eating time of control horses. Locomotion activities were unaffected by the tests, but presentation of the traffic cone and white noise elicited increased heart rates in test horses compared to control horses. Addition of eucalyptus to the food container did not elicit a heart rate response, but test horses did have an increased number of eating bouts and became more watchful towards their surroundings. Horses used for the visual and auditory tests spent more time alert towards the stimulus rather than towards their surroundings, and horses used in the auditory test took more steps backwards in response to the test. There was a correlation in the heart rate responses between tests, which reflected a non-differentiated activation of the sympathetic nervous system. Behavioural responses were linked to the type of stimulus.