Milk fat globules are the biological entities secreted by all female mammals to ensure many functions in the gastro-intestinal tract of newborns (e.g. bring energy and bioactive molecules, protection against viruses and bacteria). Milk fat globules are enveloped by a biological membrane (the Milk Fat Globule Membrane, MFGM) with a similar composition as that of the plasmatic membrane of the lactating cells (Keenan et al., 1970). However, it remains poorly known despite its importance in nutrition. Phospholipids with saturated fatty acids, and in particular milk sphingomyelin (MSM; ~30% w/w of milk phospholipids) can segregate in the plane of the MFGM and form ordered (gel or Lo) phase domains dispersed in a fluid phase of unsaturated phospholipids (Fig 1 ; Lopez et al., 2010; Murthy et al., 2015). Do these ordered phase domains create a mechanical heterogeneity in the plane of the MFGM ? AFM force spectroscopy experiments performed with model supported bilayers (2-dimensional organization), evidenced a mechanical heterogeneity in the plane of the membrane, associated with the occurrence of stiff MSM domains in the gel phase, dispersed in a continuous fluid phase of unsaturated phospholipids, such as dioleoylphosphatidycholine (DOPC) (Guyomarc’h et al., 2014). Such mechanical heterogeneity in the MFGM could have important consequences on the biological function of the milk fat globule, e.g. on digestion or on their physical stability upon processes. Our strategy is now to prepare liposomes (3-dimensional organization such as milk fat globules) and to evaluate the respective mechanical properties of gel-phase MSM and fluid-phase DOPC bilayers, using AFM. Liposomes were produced in calcium-containing aqueous environment using sonication or extrusion at 60°C, then cooled to 20°C. The morphology of the gel-phase MSM liposomes and fluid-phase DOPC liposomes were characterized by cryoTEM. For AFM experiments, the liposomes were immobilized onto flat substrates of chemically modified gold and imaged in aqueous buffer. Indentation measurements were performed at 20°C at the apex of individual liposomes (Fig. 2) and data was interpreted in terms of the respective membrane stiffness values of the MSM or DOPC curved bilayers. When applicable, a model based on the shell theory is used to infer the Young’s modulus of either type of membrane (Delorme and Fery, 2006). The results are intended to serve as reference values for further investigations on membranes containing both gel phase and fluid phase phospholipids.