Table 4.

Methodological factors affecting nutritive and non-nutritive components of human milk.

Factors	Effect on nutritive or non-nutritive components of HM	Evidence	References
Human milk expression	Hand expression compared with pump expression shows lower daily milk supply	Inconsistent or lacking	(242–247)
	Hand expression compared with pump expression results in higher sodium, potassium, proteins, total lipids	Inconsistent
Storage temperature	Higher temperatures (−20°C) reduce total lipids	Inconsistent or lacking	(248–255)
Storage length	Longer storage durations reduce vitamin C concentrations	Inconsistent or lacking	(248, 252–255)
	Longer storage durations do not affect tocopherol concentrations	Evidence probable
	Longer storage durations (12 months) reduce concentrations of IgA, IL-8, and TGF-β1	Inconsistent or lacking
Freeze-thaw cycles	Multiple cycles reduce total lipids	Evidence probable	(250, 251, 256, 257)
	Multiple cycles reduce carbohydrate concentrations	Lacking
	Multiple cycles increase lipolytic products (free FA, monoacylglycerol)	Evidence probable
Choice of analytical method	Determination of combustion in a bomb calorimeter accurately quantifies macronutrients and total energy	Evidence probable	(177, 205, 258–273)
	The Kjeldahl method or micro-Kjeldahl analysis measure true protein with high precision	Evidence probable
	High throughput spectroscopy measures protein accurately	Inconsistent
	The Folch, Bligh and Dyer and Röse-Gottlieb methods accurately measure lipid content	Evidence probable
	Spectroscopic methods determine total lipids accurately	Inconsistent