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Objective: The objective of this study is to evaluate if echocardiographic examination causes any pain response in term and preterm infants., Methods: Term and preterm neonates who admitted to Neonatal Intensive Care Unit at Gazi University Hospital and Etlik Zubeyde Hanim Training and Research Hospital and were performed echocardiography for any reason were included into the study. Neonates were evaluated before, during and 10 minutes after the examination. Vital signs (heart rate, respiratory rate, blood pressure, transcutaneous oxygen saturation) were recorded. All subjects were also evaluated with Neonatal Infant Pain Scale during the examination., Results: In this study, we evaluated 99 newborn infants. Five infants who received fentanyl treatment were excluded. The heart rate (p = 0.000), respiratory rate (p = 0.000), diastolic blood pressure (p = 0.001) and oxygen saturation (p = 0.000) during the examination were significantly different than the values before and 10 minutes after the examination. Infants whose gestational age ≤32 weeks (n:20) have significantly higher NIPS scores (mean ± SEM = 3.3 ± 0.4) than the infants whose gestational age is greater than 32 weeks (n:71) (mean ± SEM = 2.4 ± 0.2)., Conclusions: Echocardiographic examination which is known as noninvasive and painless causes significant pain in preterm infants.