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Physiological changes provide indices of stress responses, however, behavioural measures may be easier to determine. Spontaneous eye blink rate has potential as a non-invasive indicator of stress. Eyelid movements, along with heart rate (HR) and behaviour, from 33 horses were evaluated over four treatments: (1) control-horse in its normal paddock environment; (2) feed restriction-feed was withheld at regular feeding time; (3) separation-horse was removed from visual contact with their paddock mates; and (4) startle test-a ball was suddenly thrown on the ground in front of the horse. HR data was collected every five s throughout each three min test. Eyelid movements and behaviours were retrospectively determined from video recordings. A generalized linear mixed model (GLIMMIX) procedure with Sidak's multiple comparisons of least squares means demonstrated that both full blinks (16 ± 12 b vs. 15 ± 15 b vs. 13 ± 11 b vs. 26 ± 20 a full blinks/3 min ± SEM; a,b differ p &lt; 0.006) and half blinks (34 ± 15 ab vs. 27 ± 14 bc vs. 25 ± 13 c vs. 42 ± 22 a half blinks/3 min ± SEM; a,b,c differ p &lt; 0.0001) decreased during feed restriction, separation and the startle test compared to the control, respectively. Eyelid twitches occurred more frequently in feed restriction ( p &lt; 0.0001) along with an increased HR ( p &lt; 0.0001). This study demonstrates that spontaneous blink rate decreases while eyelid twitches increase when the horse experiences a stressful situation.

Eye Blink Rates and Eyelid Twitches as a Non-Invasive Measure of Stress in the Domestic Horse