It was assumed that the full-blink and half-blink rates (eye blink rate, EBR) increased in horses after stress, with these rates decreasing gradually after that. This study aimed to analyze the horses’ EBR for three minutes following exposure to a stressor. The tests were conducted on ten horses at rest /before test/ after a test involving a new object. The EBR was analyzed using Kinovea software [version 0.9.5]. Moreover, HR was measured with a stethoscope, and behavior was assessed using a 5-point scale. Compared to rest and pre-test, HR post-test increased significantly, and scores for behavior decreased during the test. There were no significant differences between EBRs in consecutive minutes at rest and pre-test. After the test, only the frequency of half-blinks and full-blinks and half-blinks together decreased significantly in the following minutes of measurement. Significant differences between EBR at rest and after the test and between EBR before and after the test occurred at each minute of the test. Only in the case of behavioral assessment and HR, the results correlated significantly. It was concluded that the analysis of EBR as a method for assessing horse stress should not be regarded as fully reliable and effective at this stage of research, as the results from this range did not correlate with the HR and the scores for behavior. In addition, it was suggested that the full-blink rate was worth using for further research.