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Background  Breast milk composition has been reported to vary significantly between individual women and between different populations. However, the composition is also known to vary within the same woman between different days, within the same day, and even across the same feed. Therefore, it is unclear to what extent variations in composition are due to variations in sampling methodology between studies. The purpose of this systematic review is to compare the results obtained for breast milk macronutrient composition between studies utilizing different sampling methodologies and to use this as a basis to determine the most robust and consistent sampling approach as an alternative to full expression (gold standard).    Methods  The EMBASE, MEDLINE/PubMed, Cochrane Library, Scopus, Web of Science, and ProQuest Dissertations and Theses Global databases will be searched for relevant articles. Observational studies, including cross-sectional, comparative cohort, and longitudinal cohort studies which involve lactating women who are breastfeeding (exclusively or not) or expressing (manually or using a breast pump) at any lactation stage will be included. This review will compare different methods of breast milk collection used in research studies which report macronutrient levels (protein, fat, lactose). Two review authors will independently screen titles and abstracts of studies identified by the literature search to determine articles for the full text screening. Quality assessment of included articles will be conducted independently by two review authors using the Newcastle-Ottawa scale.    Discussion  It is important to identify the most reliable and practical method of human milk collection which best represents the average composition of the milk that is being consumed by the infant. This systematic review will be critical for ensuring that we determine a robust and consistent sampling approach to use in future studies of evaluating breast milk composition in a larger population. Identifying a recommended standard collection protocol will also provide more opportunities for sharing and combining data from different research groups, thus enhancing replicability and knowledge in the field.    Systematic review registration  PROSPERO  CRD42017072563Electronic supplementary material  The online version of this article (10.1186/s13643-018-0788-4) contains supplementary material, which is available to authorized users.

A methodological approach to identify the most reliable human milk collection method for compositional analysis: a systematic review protocol