This study was aimed to evaluate the effects of tropicamide 0.5% eye drops on cardiovascular parameters during exercise testing. The study group included 154 healthy subjects (mean age: 44.7 ± 8 years). The subjects were divided into three groups according to the size of the pupils at the onset of exercise: A: pupils not dilated (n = 27), B: pupils partially dilated (n = 90) and C: pupils widely dilated (n = 37). They were compared to 66 healthy controls (age 43.8 ± 8) who did not receive the drops. Rest and exercise parameters were affected in groups A and B, while the results of group C resembled those of the controls: (a) resting heart rate – 66.7, 66.6, 70.9 and 69.3, respectively (p = 0.03); (b) heart rate at 50 and 100 W- 104, 107, 110 and 111 (p = 0.01) and 131, 131, 137, 139, respectively (p = 0.01); and (c) peak systolic blood pressure – 192, 186, 183, 175; respectively (p = 0.004). Reanalyzing the data by scoring of visual impairment gave identical results. As a whole, the study group achieved higher work loads than the controls (126 vs. 119 W; p = 0.03). We conclude that the instillation of ocular tropicamide has definite effects on cardiovascular parameters, both at rest and during exercise. Mainly, patients showed a lower heart rate at the initial levels of exercise. However, at symptom-limited level, tropicamide does not influence a patient’s ability to achieve the target heart rate, and stress testing results are not altered by the drug.