Background: Premature birth heightens neurodevelopmental risks, theorized to partly stem from altered sensory inputs and disrupted sleep patterns. Modifying the acoustic milieu through music intervention (MI) offers promise to improve neonatal comfort, reduce sleep disturbances, and stabilize physiological parameters. This study explores the impact of non-pharmacological MI on these health indicators within the Neonatal Intensive Care Unit (NICU)., Methods: A single-blinded RCT was conducted. Premature infants (34 > GA > 29 wks) were randomly assigned to either receive 8 min of daily MI or placebo for up to 15 days. Validated behavioral comfort scores were visually obtained by a blinded observer before and after intervention. Additionally, physiological signs (HR, RR, SatO2) were recorded. Differences between the groups were analyzed using χ² tests and t-tests., Results: In total 56 preterm infants were included. After intervention, comfort levels increased significantly in the MI compared to placebo group (p = 0.000). Neonates receiving MI transitioned from wakefulness to a state of sleep significantly more compared to placebo (p = 0.002). Physiological parameters remained stable., Conclusions: This study adds to existing literature demonstrating that a music intervention in a NICU setting can enhance comfort and sleep of premature infants without adversely affecting physiological parameters., Impact: The study demonstrates that music interventions (MI) in a Neonatal Intensive Care Unit (NICU) setting can significantly increase comfort levels and positively impact the sleep of premature infants without negatively impacting their physiological parameters. This research increases evidence for non-pharmacological interventions, specifically music, as beneficial for the well-being of premature infants in NICU settings. It replicates and expands upon previous methodological designs, providing more robust evidence of MI's positive effects on this vulnerable population. The positive outcomes of music intervention could influence hospital policies by integrating non-pharmacological practices into standard neonatal care protocols to enhance developmental support for premature infants., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)