The impact of mydriatic agents on the standardized provocation of retinal vascular reactivity has not been systematically investigated. Our aim was to investigate the effect of commonly used mydriatic agents on the provoked vascular response of retinal arterioles. One eye was randomly selected for mydriasis from 10 healthy volunteers (age 23.3 ± 4.9 years). A single drop of: 1% tropicamide (T), or a combination of 0.8% tropicamide and 5% phenylephrine (TP), or 1% cyclopentolate (C) were instilled into the volunteers lower fornix at each of three visits. Volunteers underwent a standardized isocapnic hyperoxic provocation. Four retinal hemodynamic measurements were acquired with the Canon Laser Blood Flowmeter at equivalent positions on the superior temporal arteriole (STA) and inferior temporal arteriole (ITA) at baseline, during provocation and after recovery. Statistical analysis was performed using linear mixed-effect models. Pre- and post-dilation measurements indicated that pupil diameter increased with use of T, TP, C (all <0.001), while systolic blood pressure, diastolic blood pressure and intraocular pressure did not change significantly (all >0.05). In response to a standardized isocapnic hyperoxic challenge, blood vessel diameter, blood velocity and flow decreased in both the STA and ITA relative to baseline. Comparison between the change elicited by isocapnic hyperoxic gas stimuli with respect to blood vessel diameter, blood velocity, blood flow, were equivalent for each mydriatic agent in the STA (p = 0.66, p = 0.99, p = 0.99, respectively) and the ITA (p = 0.85, p = 0.80, p = 0.66, respectively). Furthermore, comparison between the change in the STA and ITA with respect to the above parameters showed equivalent responses in both vessels for each mydriatic agent: T (p = 0.92, p = 0.99, p = 0.35; respectively), TP (p = 0.89, p = 0.96, p = 0.62; respectively), and C (p = 0.87, p = 0.35, p = 0.56; respectively). The provoked retinal vascular reactivity response to standardized isocapnic hyperoxia was equivalent irrespective of the agent used to achieve mydriasis.