Adequate nutrition of very preterm infants comprises fortification of human milk (HM), which helps to improve their nutrition and health. Standard HM fortification involves a fixed dose of a multi-nutrient HM fortifier, regardless of the composition of HM. This fortification method requires regular measurements of HM composition and has been suggested to be a more accurate fortification method. This observational study protocol is designed to assess whether the target HM fortification method (contemporary cohort) improves the energy and macronutrient intakes and the quality of growth of very preterm infants, compared with the previously used standard HM fortification (historical cohorts). In the contemporary cohort, a HM multi-nutrient fortifier and modular supplements of protein and fat are used for HM fortification, and the enteral nutrition recommendations of the European Society for Paediatric Gastroenterology Hepatology and Nutrition for preterm infants will be considered. For both cohorts, the composition of HM is assessed using the Miris Human Milk analyzer (Uppsala, Sweden). The quality of growth will be assessed by in-hospital weight, length, and head circumference growth velocities and a single measurement of adiposity (fat mass percentage and fat mass index) performed just after discharge, using the air displacement plethysmography method (Pea Pod, Cosmed, Italy). ClinicalTrials.gov registration number: NCT04400396.