The aim of this study was the analytical evaluations of human milk fat substitutes (HMFS) synthesized via enzymatic interesterification of lard and milk thistle oil mixtures by a immobilized commercial sn-1,3-specific lipase, using calorimetric and chromatographic methods. The mixtures of lard and milk thistle oil at mass ratio 6:4 and 8:2 were interesterified for 2, 4 and 6 h at the temperature of 60 °C. The determination of fatty acid composition was carried out by gas chromatographic analysis of fatty acid methyl esters. The positional distribution of fatty acids in the sn-2 and sn-1,3 positions of triacylglycerols was based on the ability of the pancreatic lipase to selectively hydrolyze ester bonds in the sn-1,3 positions. Pressure differential scanning calorimetry (PDSC) method was used for the determination of the oxidative stability of HMFS. The oxidative induction time was obtained from the PDSC curves. Due to enzymatic interesterification of mixtures of lard and milk thistle oil, new HMFS that have a similar regiospecific structure of triacylglycerols to human milk fat can be produced. The induction time obtained from PDSC measurements can be used as a parameter for the assessment of the resistance of tested fats to their thermal-oxidative decomposition.