The Science Behind Lactation Lab

The purpose of this scientific literature review is to raise awareness about how breastfeeding women with low stores or intakes of nutrients can benefit from taking supplements during lactation and provide guidance on effective doses to improve infant health. Additionally, it provides evidence based research on the maternal and infant health benefits of this strategy. The intended audience is healthcare practitioners (physicians, lactation consultants, doulas, pediatricians) who patients are breastfeeding mothers or their infants, but we hope anyone with an interest in science or medicine may find this useful. Full references are provided at the end of this review.

Why Lactation Lab

Lactation Lab empowers mothers and optimizes their children’s nutrition using a research based approach to maternal diet.

The desire for mothers and their healthcare providers to test their milk is not new. In 1978 the creamatocrit method was developed whereby a small sample (capillary tube) of breast milk is spun down in a centrifuge machine and the the layer of fat which separated from the aqueous layer is measured with a ruler and an approximate calorie count is then calculated. This method is still widely used in hospitals and breastfeeding support centers, despite 40 years of advances across the spectrum of medicine, nutritional research, lab technology and general scientific knowledge.

Lactation Lab was founded with a mission to bring scientific advancement to the field of breast milk research and for the first time ever offer a comprehensive, personalized analysis of a mother’s breast milk. Using the latest academic research and most advanced lab technologies, our team of dedicated physicians, research scientists and clinicians has truly innovated in a field that has long been neglected by science.

The Science of Breast Milk

Breast milk is a dynamic fluid that changes over time. Our research goal is to determine what changes, over what period of time and why.

Studies in the past have had challenges refining the data to the level of an individual breastfeeding mother. Older studies looked at pooled breast milk samples and did not account for the variations in diet (17,18). Some also included very small sample sizes (e.g. Hall et al. from 1979 studied 3 women). In addition, variability in the breast milk composition was often due to differing laboratory methods used for the analyses, including a) direct energy quantification by combusting in a bomb calorimetry and b) calculated energy estimates using Atwater energy multiplication factors for macronutrients such as protein, fat and carbohydrates. The methods used to measure protein also varied from a) direct quantification of the true protein content and b) quantification of the nitrogen only, which does not account for the presence of nitrogen in non-protein compounds. This has made it difficult to extract reliable data from many early studies.

The gold standard of milk collection involves sampling of the same individual over a 24-hour period when evaluating milk (19, 20) and many studies have shown that mature milk remains relatively similar in composition with subtle changes over the course of lactation (21,22). Maternal diet has a profound influence on the composition of human milk for some nutrients, (10,11,12). Breast milk varies significantly from mother to mother. We ask that mothers collect milk over a 24-hour period and not just one point in time.

Variance in milk composition for an individual mother

Protein and carbohydrates change with the overall duration of lactation but are relatively invariable between women at any stage of lactation (4)


Breast Milk from a mother who gives birth preterm will generally have a higher protein content, than that of a woman breastfeeding a toddler, since preterm infants require a higher protein diet. A study of 483 milk samples (only 20 mothers) found that protein decreased over the course of six months from 2.0 g/dl to 1.0 g/dl on average, but lactose content remained relatively constant (23, 24). A study of 15 women did not show significant changes in lactose/ carbohydrate and no difference in protein levels in samples taken over a 24 hour period (24).


Carbohydrates including lactose and human milk oligosaccharides have not been found to change significantly over time but do vary from mother to mother (23). Human milk oligosaccharides help support the gut and protect from infection (25).


Discussions about fat testing in breast milk seem to raise controversy, particularly with regard to foremilk and hindmilk.

Foremilk is typically defined as the first 3 minutes of feeding or the first 30 ml expressed from the breast. Research has shown that foremilk contains less fat that hindmilk, but one must also take into consideration that during normal feeding or pumping, the breast typically is emptied for longer than 3 minutes.

One study of 15 women found that milk ranged from 32 g/l of fat (early emptying of the breast) to 56 g/l at the end of a feed over a 24-hour period, but that during any given 24-hour period, the same child will have a consistent total fat intake (24).

A study of 71 women which collected over 775 samples of breast milk found that the fat content in milk varied over a 24-hour period and varied from mother to mother but was actually independent of the number of feeds. The study showed that foremilk fat content was not always low and “mothers can be reassured that infants who take frequent small breast feedings have the same daily fat intake as infants who wake infrequent large breastfeedings” (26).

A study of 154 milk samples from 5 women did show significant difference in total fat from foremilk and hind milk but except for fatty acids, whose levels remained stable (27).

Given the higher fat (and caloric) content of hindmilk, what could then be the role of foremilk?

A study of 30 women showed that foremilk contained a significant amount of essential amino acids when compared to hindmilk. Fatty acids are not only important building blocks for protein synthesis but optimal for infant development (28; 29; 30 and neurodevelopment (31). It is also thought that certain amino acids are essential for optimal development of the immune system and intestinal health (through T and B cell proliferation) suppression of the proinflammatory cytokine production by a variety of immune cells, and the stimulation of T cells which are important for immune function (29-30)

Mothers of babies who take small frequent feeds can sometimes be concerned that their babies are not getting enough of the hindmilk/ fat. One study looked at the variations between mother’s foremilk and hindmilk fat content depending on the number of feeds per day. Babies who took 6-9 feeds per day were compared to those whose babies took 14-18 small feeds per day. Those who ate larger less frequent meals were found to have low fat foremilk and high fat hindmilk (foremilk 4.3% to hindmilk 10.7%) when compared to mothers who took 14-18 small feeds in 24 hours those mothers and significantly more fat in the foremilk (foremilk 4.8% when compared to their hindmilk 8.2%). This study showed that there was no difference in the total fat consumed for each of these infants in 24 hours, meaning that the one who feeds frequently will have “x” amount of fat from that mom’s milk and the other baby feeding more more frequently will also consume the same “x” amount of fat from their mother over a 24-hour period. It was concluded that the baby’s fat intake over a 24-hour period is independent of the number of feeds per day (32). In other words, the baby’s fat intake was consistent from day to day.

Freshly expressed foremilk also contains lymphoid cells which play an important role in the human microbiome and immune system (33). Taking into account these critical properties of foremilk, Because of the essential amino acids and lymphoid cells present in foremilk, the fact that these are essential nutrients, it is recommended to no longer distinguish between foremilk and hindmilk. Further, because a lot of breastfeeding women choose to feed on demand (32) and studies have shown that their bodies adapt the amount of fat in the foremilk to hindmilk and that this will be consistent over a 24 hour period, we suggest moving away from these terms.

Another reason to move away from the focus on foremilk and hindmilk: It’s more that just about total fat.

Total lipid content (fat) in human milk is mostly 95-99% made up of triglycerides that contain medium chain fatty acids (c8 to C12), long chain fatty acids (C20 to C22) and vary depending on colostrum or mature milk (34). Fatty acids are important intestinal health, microbiome, and immune function. They exert an anti-microbial activity and protect the digestive tract against infections. A study of 238 healthy lactating women found that the maternal dietary intake of EPA, DHA, omega-3 and omega-6 fatty acids concentration in milk was directly related to a mother’s diet (35). This has been shown in other studies (36,37).

Polyunsaturated fatty acids (ARA arachidonic acid, LA linoleic acid, EPA eicosapentaenoic acids and DHA docosahexaenoic play important roles in infant growth and development (38, 39, 40). Some fatty acids are essential (meaning we need to consume these as our bodies don't make them (57, 63, 64).

A study of 80 mothers also found a significant relationship between the amount of ALA (alpha linoleic acid) consumed and higher concentration her milk (41). ALA is broken down to DHA in the body.

A study of 14 women showed that changing fat intake in a maternal diet resulted in a rapidly (4 day) correlated change in the concentration in their milk (42). A study of 91 breast milk samples showed a significant correlation between a mother’s diet and the fatty acid composition of human milk. They concluded that diets of women who are breastfeeding could be improved by supplementing with fatty acids (43).

DHA was also studied in a randomized trial of 10 women, where five received no supplements and the other five did over a two week period. DHA was found to have increased significantly in the breastmilk of the women supplementing, again showing a direct correlation between mother’s diet and DHA levels in breast milk (44).

A study of women in South Dakota concluded, “Offering mothers milk DHA testing and further education about the importance of DHA during lactation may advance the understanding of optimal provision for breastfeeding mothers and improve health outcomes for mothers and babies” (45).

A meta analysis looked at a total of 59 observational studies 43 interventional studies on diet and breast milk and concluded that maternal intake of fatty acids, Vitamin C, Vitamin B12, Vitamins A, E, B1, calcium, lead, mercury (46) was dependant on a mother’s diet and varied from mother to mother.

Vitamin A

Carotenoids including Vitamin A play an important role in vision, immunity, development of the nervous system and bone development. It has been well established that Vitamin A levels in milk respond to maternal supplementation and are affected by maternal diet (2,4,47). An breastfed infant's levels are directly related to dietary consumption, which in turn are dependant on a mother’s concentration (1,2,3,14, 15).

Vitamin A deficiencies in infants result in adverse outcomes including severe respiratory and gastrointestinal infections, as well as increased morbidity and mortality (5).

It is thought that human milk-borne vitamin A reduces allergy via promotion of intestinal crypt development and a reduction of gut permeability without impacting the digestion of milk (6,7).

Vitamin C

VItamin C is essential for tissue function and the formation of certain neurotransmitters as well as an essential antioxidant (63). A study of 60 healthy women taking 500 mg of Vitamin C a day resulted in higher levels in their milk (610+/- 295.5 to 716 +/- 237.5 umol/l) (48,49). Another study showed that Vitamin C in breast milk may reduce the risk of atopy in infants (50). Another study of 200 mothers also found breast milk levels to be significantly correlated with maternal diet and supplementation (51,52).

Vitamin B12

Vitamin B12 is an essential vitamin. It is naturally present in some foods and required for proper red blood cell formation, neurologic function and DNA synthesis.

A large comprehensive meta-analysis which included 59 observational studies and 43 interventional studies concluded that vitamin B12, calcium, Vitamin C were affected by maternal diet (13). Many other studies have also found a positive correlation between a mother’s nutritional status and B vitamin levels in human milk (8,9,53,54,55).


Iron is the essential component of hemoglobin an erythrocyte protein that transfers oxygen from the lungs to the tissues. A large randomized study of 360 women who were divided into two groups: a control group receiving a placebo tablet and another group that received 200 mg of iron tablets a day. The mothers receiving the iron supplements had higher levels of iron in their milk (56).

Another study showed that iron content in breast milk is impaired when mothers have iron-de?ciency during the pregnancy period. Even if mothers had anaemia during pregnancy, our data shows that a program that assures a personalized iron treatment, improves breast milk iron content (58,59).

Among lactating women, iron deficiency has the same effects as on non-pregnant, non-lactating women of reproductive age: increased risk of iron deficiency anemia, reduced work and mental capacity, increased risk of postpartum depression and other emotional disorders, as well as reduced quality of mother-child interactions (60).


Calcium is necessary for neurotransmitter function, muscle contraction, enzyme cofactors and many other essential biochemical and physiological processes. A 2013, study of 27 women showed that calcium concentration in breast milk was significantly affected by dietary intake (61).

We do not test for Vitamin D, zinc, phosphorus, sodium, or selenium because research that not shown that these levels are affected by maternal diet.


Note that if you do not have a PubMed subscription, accessing journal articles may be difficult or expensive. We suggest seeing if your local university provides PubMed access to the public (many do). In some cases, if you contact the author directly, they will be happy to email you their research for free.

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7. Turfkruyer M., Rekima A., Macchiaverni P., Le Bourhis L., Muncan V., van den Brink G.R., Tulic M.K., Verhasselt V. Oral tolerance is inefficient in neonatal mice due to a physiological vitamin a deficiency. Mucosal Immunol. 2016;9:479–491.

8. Duggan C., Srinivasan K., Thomas T., Samuel T., Rajendran R., Muthayya S., Finkelstein J.L., Lukose A., Fawzi W., Allen L.H., et al. Vitamin B12 supplementation during pregnancy and early lactation increases maternal, breast milk, and infant measures of vitamin b-12 status. J. Nutr. 2014;144:758–764.

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24. Khan, S., Hepworth, A. R., Prime, D. K., Lai, C. T., Trengove, N. J., & Hartmann, P. E. (2013). Variation in Fat, Lactose, and Protein Composition in Breast Milk over 24 Hours: Associations with Infant Feeding Patterns. Journal of Human Lactation, 29(1), 81–89

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