•Investigation of acute stress-induced changes in percentage of visible eye white and maximum eye temperature by using a cattle crush as stressor and a feeding situation as control treatment.•Cattle crush situation was indeed stressful as seen in salivary cortisol and heart beat parameters.•No effect of cattle crush treatment on visible eye white or maximum eye temperature.•Breed-specific differences in visible eye white and maximum eye temperature. The aim of this study was to investigate if visible eye white and eye temperature measurements are feasible non-invasive physiological indicators of acute stress in cows when they are exposed to cattle crush treatment for claw trimming. In the experimental setting, 30 cows of two breeds (Red Holstein and Brown Swiss) were exposed to a non-stressful (feeding) and a stressful situation (claw trimming in a cattle crush) for 10min each. We took pictures of the eyes at 0, 5 and 10min after starting of exposure to measure the percentage of visible eye white (from photographs) and the maximum eye temperature (from thermographs). Heart rate and heart rate variability parameters were recorded continuously throughout both situations. Twenty minutes after the beginning of each situation, saliva samples were taken to determine the cortisol concentration. As expected, sympathetic activity and cortisol concentration were higher in the claw trimming than in the feeding situation. However, neither maximum eye temperature nor percentage of visible eye white differed between treatments. Instead, the results of these measurements differed between the breeds. The maximum eye temperature increased during and after both situations in Brown Swiss cows, whereas in Red Holstein cows, it increased after (but not during) both situations. Furthermore, we found that Red Holstein cows had a lower percentage of visible eye white than Brown Swiss in general. This finding might be due to differences in eye coloration patterns, with Red Holstein cows having more contrast between eye white and iris and Brown Swiss having less contrast because of their darker eye white. This breed effect might have masked potential treatment effects.