This article presents the study protocol for a randomized controlled trial (RCT) investigating the impact of singing on the brain activity of premature infants in the Neonatal Intensive Care Unit (NICU). The study focuses on how the differentiation of voices, as defined by the fundamental frequency (F0) shaped by biological sex and kinship, influences neurophysiological responses when measured by electroencephalography (EEG). Premature infants, who are highly sensitive to auditory stimuli, may benefit from music-based interventions; however, there is limited understanding of how voice variations between male and female caregivers, and whether they are biologically related, affect brain activity. Our protocol outlines a structured intervention where infants are exposed to singing by four facilitators - a male music therapist, a female music therapist, the mother, and the father - and includes two singing stages: a sustained note (A at 440 Hz) and a 90-second lullaby, both interspersed with silent periods to allow for baseline measurements. EEG recordings track brain activity throughout these sessions, followed by quantitative EEG (qEEG) analysis and thorough statistical computations (e.g., mixed-effects models, spectral power analysis, and post-hoc tests) to explore how these auditory stimuli influence brain function. Preliminary data from five infants show that maternal singing elicits the highest delta spectral power in all measured conditions except during the ‘lullaby song’, where paternal singing elicits the highest effects followed by the male music therapist and then the mother. These early findings highlight the potential influence of parental voices, particularly the fathers’ voice, on neonatal brain development, while the detailed study protocol ensures rigor and replicability, providing a robust framework for future research. Additionally, this protocol lays the groundwork for exploring the long-term effects of music-based interventions, with the goal of improving neurodevelopmental outcomes in premature infants through tailored auditory stimulation. (clinincaltrials.gov unique identifier: NCT06398912)

The authors have declared no competing interest.

clinincaltrials.gov unique identifier: NCT06398912

The author(s) received no specific funding for this work.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The research protocol has been thoroughly reviewed and approved by the ‘Panagiotis and Aglaia Kyriakou’ Hospital's Scientific Council , under the Institutional Review Board (IRB) with protocol number 11333, 09th/04-05-2023 (Θ: 9). Informed consent to participate in this study was obtained both orally and in writing.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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Data cannot be shared publicly because of the infant population included in the measurement process. Data are available from the Director of the NICU or the Lead Author of the manuscript for researchers who meet the criteria for access to confidential data.