MILQ Study Provides Reference Values for Human Milk

Written by: Lauren Milligan Newmark, Ph.D. | Issue # 130 | 2026

The Mothers, Infants, and Lactation Quality (MILQ) study had an ambitious goal—to establish accurate and globally representative reference values (RVs) for human milk macro- and micronutrients over the first 8.5 months of lactation. 

Official nutrient intake recommendations for infants and lactating women depend on human milk RVs. And, just like Dietary Reference Intakes (DRIs) for children and adults, human milk RVs must be evidence-based and derived from a large, diverse data set. However, previous studies on human milk differed in their methods of milk collection and analysis. Many failed to control for whether mothers were supplemented or well nourished, and sample sizes were often too small to be representative of a population [1-3]. Despite nearly 75 years of research on human milk composition, many official nutrient recommendations for infants and lactating mothers were based on surprisingly incomplete or unreliable data sets [2]. 

“[Researchers] did as good as they could with what they had,” said Dr. Lindsey Allen, a Research Scientist at the USDA ARS Western Human Nutrition Research Center and member of the MILQ study team. “But there were just a lot of nutrients we didn’t have values for, or the data were messy and unreliable.” 

Allen, a leading expert on micronutrient deficiencies, was particularly frustrated by the lack of sufficient data on essential micronutrients like B12 and B2 (riboflavin) [4, 5]. “Riboflavin recommendations were based on milk samples from only five women, and B12 on samples from nine women,” she shares. “These aren’t representative samples.” 

Without knowing what adequate nutrient levels in human milk are for these vitamins, it is not possible for researchers, like Allen, to make recommendations for supplementing mothers or infants. Human milk is considered the gold standard for infant nutrition, but what does “gold” mean for each nutrient?

Determined to solve this issue, Allen and an international team of collaborators formed the MILQ consortium and decided to mine for liquid gold, measuring milk composition and milk volume (infant intake) from over 1000 well-nourished mothers from four global populations (Bangladesh, Brazil, Denmark, and The Gambia) over the first 8.5 months of lactation. 

To be confident in their results, the study design needed to be meticulous. Before they even enrolled their first mother or collected their first milk sample, the MILQ team had to establish analytical methods that could accurately quantify each milk macro- and micronutrient. 

“We had to recognize that the milk matrix is very different than the plasma matrix,” Allen explains. Many previous studies used blood-based analytics to measure milk components, but lab methods must be validated for milk and account for the ways nutrients can interact within the milk matrix. “We had to make sure that if we were measuring a nutrient, there wasn’t interference from another milk component.” For example, much of human milk B12 is bound to a protein, so accurately measuring milk B12 requires first separating the vitamin from this protein [6]. For this critical step, Allen credits her collaborator Dr. Daniela Hampel, a Project Scientist in the Department of Nutrition at University of California, Davis. “[Dr. Hampel] spent 10 years perfecting the methods to analyze milk samples using internal standards and methods of validation. Without this painstaking work, this project couldn’t have happened.”

Part of this painstaking work involved validating an analytical tool called liquid chromatography tandem mass spectrometry. This tool allows for the simultaneous measurement of multiple micronutrients from a small sample (1 milliliter) of milk, even when those nutrients are present in trace amounts. The liquid chromatography phase separates the different vitamins present in the sample based on how quickly they travel through a column. Then, the mass spectrometer identifies and quantities the concentration of each vitamin present. This method proved particularly useful for the sensitive quantification of vitamin D, B vitamins, and minerals [1, 3, 7]. 

With their analytical methods established, the MILQ researchers underwent the Herculean task of recruiting over 1000 mothers, collecting milk, serum and stool samples, and measuring milk intake across multiple time points between birth and 8.5 months [3]. Data collection was rigorously uniform across sites, and, combined with the specific and objective inclusion criteria—focusing on well-nourished mothers who were not taking any supplements—ensured that their data could be reliably compared between and within study sites [1]. 

After five years of data collection and several more years of data analysis, the results were published [3, 4, 7-10]. They clearly demonstrate why this study was so imperative for understanding infant nutrient requirements. For many nutrients, particularly the infrequently studied micronutrients, the median concentrations identified by the MILQ study were lower—some by as much as 80%— than the current targets provided by the National Academy of Medicine (previously known as the Institute of Medicine, or IOM) [4, 7, 9, 10]. 

As an example, vitamin D levels were 60 -80% lower than the estimates previously used by the IOM [9]. Human milk is known to be low in vitamin D; lower, in fact, than the amount infants need for optimal growth and development [11]. The MILQ study finding that the median concentration of milk vitamin D was significantly lower than IOM recommendations, even in populations with year-round ultra-violet light exposure like The Gambia, Bangladesh, and Brazil, suggests that infant vitamin D supplementation may be necessary for most infants, regardless of maternal nutrition status. For this nutrient, it is not only a dietary issue. Cultural practices, like clothing, sunscreen, and spending daylight hours inside can limit ultra-violet light exposure and prevent vitamin D production in both the mother and the infant. 

“The differences with the IOM are probably due to our standardized methods, our improvements in methods, [and] looking at milk in larger populations and in multiple places,” explains Allen. “One milk sample is not likely to be representative of milk composition over the course of lactation,” explains Allen. “You have to look at a lot of samples from a lot of women, and we know you have to look across lactation.” 

Similarly, there isn’t one value for any nutrient that equates to the “gold standard.” Rather, MILQ RVs represent the “acceptable range of variation in a healthy and diverse population of lactating women” [12]. Much like pediatricians use growth charts to determine if a child is following a normal, healthy growth pattern, human milk RVs have an upper and lower limit that can be used to identify populations where milk nutrient concentrations fall below or above these RV percentiles and therefore may be at risk of deficiency. Moreover, they provide a critical resource to evaluate the impact of nutrition interventions [3, 12]. 

“We have percentile curves for each nutrient across the first 8.5 months of lactation, and we can see what was the average percentile before intervention and what is the change in the percentile after intervention,” says Allen. “We hope to use this data to guide multiple micronutrient supplements during pregnancy and lactation to improve infant outcomes.” 

The MILQ study is the most comprehensive study of its kind [1], but they aren’t done yet. “There is a huge amount of information that we still want to get from our milk samples,” says Allen. “What can we do that no one has done before?” 

One novel thing they are doing is taking a systems biology approach to understanding human milk. “We are doing multi -omics studies…proteomics, metabolomics, genomics.” This approach means that they are measuring everything they can in each milk sample. From improved methods to identify and measure human milk oligosaccharides, to utilizing milk and fecal sample data to investigate the influence of human milk on the infant gut microbiome, the next stages of the MILQ study will transform our understanding of how human milk feeding optimizes infant health and development. 

References

1. Bremer AA. A significant step forward in understanding human milk: the mothers, infants, and lactation quality study. Advances in Nutrition. 2025 Oct 1; 16.
2. Allen LH, Hampel D, Shahab-Ferdows S, Andersson M, Barros E, Doel AM, Eriksen KG, Christensen SH, Islam M, Kac G, Keya FK. The Mothers, infants, and lactation quality (MILQ) study: a multi-center collaboration. Current Developments in Nutrition. 2021 Oct 1;5(10): nzab116.
3. Allen LH, Moore SE, Kac G, Michaelsen KF, Mølgaard C, Islam MM, Shahab-Ferdows S, Christensen SH, Lewis JI, Peerson JM, Tan X. The Mothers, infants, and lactation quality (MILQ) study: introduction and study design. Advances in Nutrition. 2025 Oct 1;16: 100499.
4. Allen LH, Shahab-Ferdows S, Moore SE, Peerson JM, Kac G, Figueiredo AC, Dror DK, Michaelsen KF, Islam MM, Nije F, Hampel D. Reference values for B vitamins in human milk: the mothers, infants, and lactation quality (MILQ) study: Advances in Nutrition. 2025 Oct 1;16: 100500.
5. Allen LH, Dror DK. Introduction to current knowledge on micronutrients in human milk: adequacy, analysis, and need for research. Advances in Nutrition. 2018 May 1;9: 275S-7S.
6. Hampel D, Shahab-Ferdows S, Domek JM, Siddiqua T, Raqib R, Allen LH. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk. Food chemistry. 2014 Jun 15; 153:60-5.
7. Allen LH, Islam MM, Kac G, Michaelsen KF, Moore SE, Andersson M, Peerson JM, Doel AM, Dror DK, Shahab-Ferdows S, de Barros Mucci D. Reference values for minerals in human milk: the mothers, infants and lactation quality (MILQ) study. Advances in Nutrition. 2025 Aug 26:100431.
8. Moore SE, Devi S, Kurpad A, Peerson JM, Christensen SH, Islam MM, Kac G, Michaelsen KF, Silva GT, Allen LH, Mølgaard C. Breast milk Intake from 1 to 8.5 months of lactation in the multisite Mothers, Infants and Lactation Quality (MILQ) study. Advances in Nutrition. 2025 Aug 26:100456.
9. Kac G, Jones KS, Meadows SR, Hampel D, Islam MM, Mølgaard C, Moore SE, Dror DK, Shahab-Ferdows S, de Barros Mucci D, Figueiredo AC. Reference values for fat-soluble vitamins in human milk: the mothers, infants and lactation quality (MILQ) study. Advances in Nutrition. 2025 Aug 26:100484.
10. Lewis JI, Dror DK, Hampel D, Kac G, Mølgaard C, Moore SE, Peerson JM, Christensen SH, Islam MM, de Barros Mucci D, Figueiredo AC. Reference values for macronutrients in human milk: the mothers, infants and lactation quality (MILQ) study. Advances in Nutrition. 2025 Oct 1;16: 100501.
11. Gartner, L.M. and Greer, F.R. 2003. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics, 111(4): 908-910.
12. Moore SE, Allen LH, Kac G, Michaelsen KF, Mølgaard C, Islam MM, Andersson M, Shahab-Ferdows S, Christensen SH, Lewis JI, Peerson JM. Future applications of human milk reference values for nutrients: a global resource for maternal and child nutrition research. Advances in Nutrition. 2025 Aug 26: 100429.
13. Christian P, Smith ER, Lee SE, Vargas AJ, Bremer AA, Raiten DJ. The need to study human milk as a biological system. The American Journal of Clinical Nutrition. 2021 May 1;113(5): 1063-72.

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