Aims: This study was conducted to determine the effects of Facilitated Tucking (FT) with hands or a positioning bed on cerebral oxygenation (rSO2), physiological parameters, pain and comfort of premature infants during heel blood collection. Methods: This prospective, single-blinded, randomized controlled trial was conducted in the tertiary neonatal intensive care unit of the Faculty of Medicine Hospital in Türkiye between December 2020 and October 2021. Heel blood collection was performed in the facilitated tucking with hands group (n = 22), facilitated tucking with positioning bed group (n = 22) and routine procedure control group (n = 22); rSO2, physiological parameters, pain and (dis)comfort were assessed. Results: In the facilitated tucking with positioning bed group, rSO2 and oxygen saturation (SpO2) were significantly higher during and after the procedure compared with the other two groups, while respiratory rate and pain score were significantly lower and (dis)comfort level was significantly higher. In addition, heart rate during the procedure was significantly lower in the facilitated tucking with positioning bed group than in the other two groups (p < 0.001). According to the results of the analysis, the effect size was moderate for rSO2, body temperature, heart rate and SpO2 and large for respiratory rate, comfort and pain. Conclusions: Positioning bed can help stabilize rSO2 and physiologic parameters, increase comfort and reduce pain in premature infants.