Comparison of different protein concentrations of human milk fortifier for promoting growth and neurological development in preterm infants

Review question

Among preterm infants, does the amount of protein used to fortify breast milk feeds result in any difference in growth and neurodevelopmental (development of the brain to improve performance or functioning (e.g. intelligence, reading ability, social skills, memory, attention or focus skills) outcomes?


Breast milk is the best form of nutrition for preterm infants. However, as preterm infants often have difficulties tolerating large amounts of milk, they may not get the recommended amounts of protein from breast milk alone. It has become common clinical practice to fortify breast milk for preterm infants with additional nutrients with a product known as human milk fortifier. Over time the amount (concentration) of protein used in human milk fortifiers has increased, but there is debate about what the optimal protein concentration of human milk fortifier is.

Study characteristics

This review included nine trials involving 861 infants. Six trials compared a high versus moderate concentration of protein in the human milk fortifier, and three trials compared a moderate versus low concentration of protein. Our main outcomes were growth (e.g. weight, length, head circumference), neurodevelopment and death. Reporting was incomplete for all outcomes; most were at low or unclear risk of bias. The search is up to date as of August 2019.

Key results

Feeding preterm infants with a human milk fortifier that contains a high protein concentration versus a moderate protein concentration resulted in small increases in weight gain but not length gain or head growth during hospital admission after birth. There were small increases in weight gain and length gain in infants fed a human milk fortifier that contained moderate concentrations of protein compared with low concentrations. There was no clear effect of protein concentration on infant death during the initial hospitalisation. There were only limited data on other health outcomes, and this evidence suggests the amount of protein in human milk fortifier does not affect the risk of infections or feeding or bowel problems. There were no available trial data about infant growth after hospital discharge, or long term development.


Although there was some evidence that use of human milk fortifiers with a high or moderate protein concentration are associated with small increases in weight gain during hospital stay, there are no data about the impact on growth after the hospital admission or on developmental outcomes. Further well designed trials are needed to determine whether the amount of protein in human milk fortifiers is associated with benefits or harms in the longer term.

Certainty of the evidence

The certainty of this evidence was very low to moderate due to inconsistent results reported by some trials and potential bias related to the way some trials were conducted.

Authors' conclusions: 

Feeding preterm infants with a human milk fortifier containing high amounts of protein (≥ 1.4g/100 mL EBM) compared with a fortifier containing moderate protein concentration (≥ 1 g to < 1.4 g/100 mL EBM) results in small increases in weight gain during the neonatal admission. There may also be small increases in weight and length gain when infants are fed a fortifier containing moderate versus low protein concentration (< 1 g protein/100 mL EBM). The certainty of this evidence is very low to moderate; therefore, results may change when the findings of ongoing studies are available. There is insufficient evidence to assess the impact of protein concentration on adverse effects or long term outcomes such as neurodevelopment. Further trials are needed to determine whether modest increases in weight gain observed with higher protein concentration fortifiers are associated with benefits or harms to long term growth and neurodevelopment.

Read the full abstract...

Human milk alone may provide inadequate amounts of protein to meet the growth requirements of preterm infants because of restrictions in the amount of fluid they can tolerate. It has become common practice to feed preterm infants with breast milk fortified with protein and other nutrients but there is debate about the optimal concentration of protein in commercially available fortifiers.


To compare the effects of different protein concentrations in human milk fortifier, fed to preterm infants, on growth and neurodevelopment.

Search strategy: 

We used the standard search strategy of Cochrane Neonatal to search CENTRAL (2019, Issue 8), Ovid MEDLINE and CINAHL on 15 August 2019. We also searched clinical trials databases and the reference lists of retrieved articles for randomised controlled trials and quasi-randomised trials.

Selection criteria: 

We included all published and unpublished randomised, quasi-randomised and cluster-randomised trials comparing two different concentrations of protein in human milk fortifier.

We included preterm infants (less than 37 weeks' gestational age). Participants may have been exclusively fed human milk or have been supplemented with formula.

The concentration of protein was classified as low (< 1g protein/100 mL expressed breast milk (EBM)), moderate (≥ 1g to < 1.4g protein/100 mL EBM) or high (≥ 1.4g protein/100 mL EBM). We excluded trials that compared two protein concentrations that fell within the same category.

Data collection and analysis: 

We undertook data collection and analyses using the standard methods of Cochrane Neonatal. Two review authors independently evaluated trials. Primary outcomes included growth, neurodevelopmental outcome and mortality. Data were synthesised using risk ratios (RR), risk differences and mean differences (MD), with 95% confidence intervals (CI). We used the GRADE approach to assess the certainty of the evidence.

Main results: 

We identified nine trials involving 861 infants. There is one trial awaiting classification, and nine ongoing trials. The trials were mostly conducted in infants born < 32 weeks' gestational age or < 1500 g birthweight, or both. All used a fortifier derived from bovine milk. Two trials fed infants exclusively with mother's own milk, three trials gave supplementary feeds with donor human milk and four trials supplemented with preterm infant formula. Overall, trials were small but generally at low or unclear risk of bias.

High versus moderate protein concentration of human milk fortifier

There was moderate certainty evidence that a high protein concentration likely increased in-hospital weight gain compared to moderate concentration of human milk fortifier (MD 0.66 g/kg/day, 95% CI 0.51 to 0.82; trials = 6, participants = 606). The evidence was very uncertain about the effect of high versus moderate protein concentration on length gain (MD 0.01 cm/week, 95% CI –0.01 to 0.03; trials = 5, participants = 547; very low certainty evidence) and head circumference gain (MD 0.00 cm/week, 95% CI –0.01 to 0.02; trials = 5, participants = 549; very low certainty evidence).

Only one trial reported neonatal mortality, with no deaths in either group (participants = 45).

Moderate versus low protein concentration of human milk fortifier

A moderate versus low protein concentration fortifier may increase weight gain (MD 2.08 g/kg/day, 95% CI 0.38 to 3.77; trials = 2, participants = 176; very low certainty evidence) with little to no effect on head circumference gain (MD 0.13 cm/week, 95% CI 0.00 to 0.26; I² = 85%; trials = 3, participants = 217; very low certainty evidence), but the evidence is very uncertain. There was low certainty evidence that a moderate protein concentration may increase length gain (MD 0.09 cm/week, 95% CI 0.05 to 0.14; trials = 3, participants = 217).

Only one trial reported mortality and found no difference between groups (RR 0.48, 95% CI 0.05 to 5.17; participants = 112).

No trials reported long term growth or neurodevelopmental outcomes including cerebral palsy and developmental delay.