The aim of the present study was to examine the efficacy and potential side effects of repeated doses of oral sucrose for pain relief during procedures in NICU. Thirty-three preterm neonates were randomly allocated in blind fashion into two groups, the sucrose group (SG = 17) and the control group (CG = 16). The responses of neonates to pain and distress were assessed during blood collection on four consecutive assessment (ass.) days. For the first assessment, the neonates did not receive any solution before the blood collection procedure. During the next three days, the SG received oral sucrose (25%; 0.5 ml/kg) and the CG received sterile water, 2 min before each minor acute painful procedure. The neonates were evaluated during blood collection each morning. The assessment was divided into five phases: Baseline (BL), Antisepsis (A), Puncture (P), Dressing (D), and Recovery (R). The neonates’ facial activity (NFCS), behavioral state, and heart rate were evaluated. The data analysis used cut-off scores for painful and distressful responses. No side effects of using sucrose were detected. There were significantly fewer SG neonates with facial actions signaling pain than CG neonates in P (ass.2) and in A (ass.3). We found significantly fewer SG neonates in the awake state than CG neonates in P (ass.2 and ass.4). There were significantly fewer SG neonates crying during A (ass.2), P (ass.2 and ass.4), and D (ass.3). There was no statistical difference between-groups for physiological response. The efficacy of sucrose was maintained for pain relief in preterm neonates with no side effects.