INTRODUCTION: The gut-brain axis plays a crucial role in the regulation and development of psychological and physical processes. The first year of life is a critical period for the development of the gut microbiome, which parallels important milestones in establishing sleep rhythm and brain development. Growing evidence suggests that the gut microbiome influences sleep, cognition and early neurodevelopment. For term-born and preterm-born infants, difficulties in sleep regulation may have consequences on health. Identifying effective interventions on the gut-brain axis in early life is likely to have long-term implications for the health and development of at-risk infants. METHODS AND ANALYSES: In this multicentre, four-group, double-blinded, placebo (PLC)-controlled randomised trial with a factorial design, 120 preterm-born and 260 term-born infants will be included. The study will investigate whether the administration of daily synbiotics or PLC for a duration of 3 months improves sleep patterns and neurodevelopmental outcomes up to 2 years of age. The trial will also: (1) determine the association between gut microbiota, sleep patterns and health outcomes in children up to 2 years of age; and (2) leverage the interactions between gut microbiota, brain and sleep to develop new intervention strategies for at-risk infants. ETHICS AND DISSEMINATION: The NapBiome trial has received ethical approval by the Committee of Northwestern and Central Switzerland and Canton Vaud, Switzerland (#2024-01681). Outcomes will be disseminated through publication and will be presented at scientific conferences. Metagenomic data will be shared through the European Nucleotide Archive. TRIAL REGISTRATION NUMBER: The US National Institutes of Health NCT06396689.