IMPORTANCE Protein, supplied in currently available commercial fortifiers, may be inadequate to meet the requirements of very preterm infants; in addition, intraindividual and interindividual variability of human milk protein and energy content potentially contribute to unsatisfactory early postnatal growth. OBJECTIVE To determine effects on growth of different levels of enteral protein supplementation in predominantly human milk-fed preterm infants. DESIGN, SETTING, AND PARTICIPANTS This randomized clinical and partially blinded single-center trial was conducted in a neonatal tertiary referral center in Germany. Sixty preterm infants (gestation <32 weeks and weight <1500 g at birth) were recruited from October 2012 to October 2014 and included 35% of 173 eligible infants. Median (interquartile range [IQR]) gestational age at birth was 29.9 (28.7-31.2) weeks. All analyses were conducted in an intention-to-treat population. INTERVENTIONS Infants were randomly assigned to either a lower-protein (adding1g of bovine protein/100mLof breast milk through a commercial human milk fortifier; n = 30) or a higher protein group at a median (IQR) postnatal age of 7 (6-8) days. The higher-protein group (n = 30) received either standardized higher-protein supplementation (study fortifier adding 1.8 g of bovine protein/100mLof breast milk [n = 15]) or individualized high-protein supplementation based on protein and fat content of administered breast milk (n = 15). Study interventions were continued for a median (IQR) of 41 (30-57) days and until definite discharge planning. MAIN OUTCOMES AND MEASURES Primary outcome was weight gain (g/kg/d) from birth to the end of intervention. RESULTS Sixty preterm infants (gestation <32weeks andweight < 1500 g at birth), 33 girls, were recruited from October 2012 to October 2014 and included 35% of 173 eligible infants. Median (IQR) gestational age at birth was 29.9 (28.7-31.2) weeks. Demographic characteristics and hospital courses were similar in both groups, and birth weights ranged from 580 to 1495 g in the lower-protein group and 490 to 1470g in the higher-protein group. Weight gain was similar in the lower-and higher-protein groups: mean (95% CI), 16.3 g/kg/d (15.4-17.1 g/kg/d) in the lower protein group vs 16.0g/kg/d (15.1-16.9 g/kg/d) in the higher-protein group) (P =.70), despite an increase in actual protein intake by0.6 g/kg/d (0.4-0.7 g/kg/d) (P <.001). Head circumference and lower leg longitudinal growth were also similar, as was the proportion of cumulative total enteral feeding volume provided as breast milk: median (IQR) proportion of breast milk, 92% (79%-98%) in the lower-protein group vs 94%(62%-99%) in the higher-protein group (P =.89). CONCLUSIONS AND RELEVANCE An increase in protein intake by 0.6 g/kg/d to a mean intake of 4.3 g/kg/d did not further enhance growth of very preterm infants with a median birth weight of 1200 g, who achieved near-fetal growth rates. This might point to a ceiling effect for enteral protein intake with respect to its influence on growth.