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To determine whether adverse effects manifested via vital sign changes during the screening examination for retinopathy of prematurity (ROP) are due to the pharmacologic properties of the eye drops or to physical manipulation of the eyes. The authors also investigated the relationship between distress during the screening process and the severity of prematurity of the infant.A prospective observational study was designed that enrolled all infants either weighing ≤1500 g or who were ≤32 weeks gestational age at birth who were admitted to the neonatal intensive care unit (NICU) at Northwest Texas Hospital or Baptist St. Anthony’s Hospital from June 2002 to February 2003. Thirty participants were enrolled in this study. Blood pressure, pulse, temperature, respiratory rate, and O2 saturation were recorded at different time intervals during the examination. Infants were excluded from the study if they were on the ventilator, considered acutely ill, born with significant birth defects, or currently taking inotropic drugs, or had received albuterol 2 hours before the examination.Oxygen saturation and pulse rate following physical manipulation of the eyes significantly varied from baseline values and the values obtained during the three instillations of topical mydriatics. No significant changes in blood pressure, temperature, or respiratory rate from their respective baseline values were observed throughout the ROP screening examination. Gestational age of the infant did not correlate with level of distress during the examination.Regardless of the severity of prematurity, infants seem to undergo significant distress during the eyelid speculum examination. Thus ophthalmologists should take into consideration the infant’s discomfort caused by physical manipulation of the eyes and attempt to perform the examination as swiftly, yet safely, as possible using topical anesthetic.