Osteopathic manipulative treatment (OMT) has been found to be effective in the context of premature infants. Nonetheless, no studies have investigated the immediate effects of OMT on heart rate variability (HRV). As altered HRV reflects poor or worsening newborn’s clinical conditions and neurodevelopment, should OMT improve HRV fluctuations, it could become a relevant intervention for improving the care of preterm newborns. Therefore, this study aimed to evaluate whether OMT could affect HRV. The study was carried out at the Buzzi Hospital in Milan. From the neonatal intensive care unit, ninety-six preterm infants (41 males) were enrolled and were randomly assigned to one of two treatment groups: OMT or Static Touch. The infants were born at 33.5 weeks (±4.3) and had a mean birth weight of 2067 g (±929). The study had as primary outcome the change in the beat-to-beat variance in heart rate measured through root mean square of consecutive RR interval differences (RMSSD); other metrics were used as secondary and exploratory analyses. Despite the lack of statistically significant results regarding the primary outcomeand some study limitations, compared to static touch, OMT seemed to favor a parasympathetic modulation and improved HRV, which could reflect improvement in newborn’s clinical conditions and development.