Effects of Fortified Formula Milk Supplementation on Neurocognitive Development and the Microbiota–Gut–Brain Axis in Preschool Children: A Cluster-Randomized, Double-Blind, Controlled Trial

Background/Objectives: The preschool period is critical for neurodevelopment, yet evidence investigating fortified formula’s effect and potential microbiota–gut–brain axis mechanisms in this age group is limited. To evaluate fortified formula milk’s effect on neurodevelopment and explore potential microbiota–gut–brain axis mechanisms in preschool children. Methods: In this 9-month cluster-randomized, double-blind, controlled trial, 120 healthy children aged 3–6 years from four kindergarten classes were stratified by grade and randomly allocated (1:1) to receive either multi-nutrient fortified formula (intervention, n = 60) or standard control milk (n = 60). Neurocognitive function was assessed using the Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition (WPPSI-IV). Safety was evaluated through anthropometry and blood biochemistry. Gut microbiota (16S rRNA sequencing) and fecal metabolomes (untargeted LC-MS) were analyzed at baseline and 9 months. Results: The intention-to-treat (ITT) analysis showed no significant difference in Full Scale Intelligence Quotient (adjusted mean difference: 1.05 points; 95% CI: −1.42, 3.52; p = 0.400). However, the intervention group significantly improved the Processing Speed Index (adjusted mean difference: 5.91 points; 95% CI: 1.88, 9.93; p = 0.004), increased gut microbial alpha diversity (Shannon index) and Bifidobacterium abundance. Metabolomic analysis revealed elevated fecal 2-hydroxybutyric acid (2-HB), a marker of propanoate metabolism. Increases in both Bifidobacterium and 2-HB levels showed a positive association with PSI improvement (both p < 0.05). All children maintained normal growth and safety parameters. Conclusions: Fortified formula milk improved processing speed in preschoolers, a benefit associated with gut ecosystem modulation characterized by Bifidobacterium enrichment and upregulated microbial propanoate metabolism. These results offer preliminary evidence for the role of the microbiota–gut–brain axis in nutritional cognitive programming during early childhood. (Clinical Trial Registry: ChiCTR2400084211).