The stress reaction of trainees is an issue in the practices of medical-related examinations that involve real-time decision making based on the examiner–subject interactions. The Goldmann perimetry (GP) test is one of these examinations. To evaluate the students’ stress reactions in the practice of the GP test, the stress-related substances and heart rate variability were measured in forty students enrolled in the practice. While there was no significant increase in stress-related substances during the practice, significantly increased sympathetic activities were observed at the beginning of the tests. Moreover, the plasma cortisol measured before the tests showed a significant positive correlation to the students’ self-confidence scores, indicating the students, especially those with higher self-confidence scores, were anxious for upcoming tests with unfamiliar subjects. Once the tests began, they felt relieved in the procedures they had learned repeatedly. On the other hand, while the average plasma ACTH decreased significantly during the test, the ACTH secretion correlated positively to the test duration, indicating that the skillful participants had less stress during the test. In the medical-related practices, pre-training on how to deal with unfamiliar subjects may be helpful for reducing the stress of the trainees, in addition to the procedure itself.