Measuring animal stress is fundamentally important for assessing animal emotional state and welfare. Conventional methods of quantifying stress (cortisol levels, heart rate/heart rate variability) require specialist equipment and are not instantly available. Spontaneous blink rate (SBR) has previously been used to measure stress responses in humans and may provide a non-invasive method for measuring stress in other animal species. Here we investigated the use of SBR as a measure of stress in the domestic horse. SBR was measured before and during a low-stress event (sham clipping) and compared with heart rate variability and salivary cortisol. For the entire sample, there was a reduction in SBR (startle response) during the first minute of clipping. For horses reactive to clipping, the initial reduction in SBR was followed by an increase above baseline whereas the SBR of the non-reactive horses quickly returned to baseline. For the entire sample, SBR correlated with heart rate variability and salivary cortisol. We have demonstrated that SBR is a valid fast alternative measure of stress in horses, but the initial 'startle' response must be considered when using this parameter as a measure of animal stress.