The aim of the study was evaluation of the possibility of ESD phenotyping in simulated casework bloodstains in relation to the temperature of storage and to the type and character of substrate. Bloodstained materials were exposed to extreme temperature conditions including -20 degrees C, 4 degrees C, 22 degrees C and 45 degrees C and subsequently phenotyped with the use of electrophoresis and isoelectric focusing. Presented results confirm faster decrease of the enzyme total activity in the conditions of increased temperature manifesting with relatively lower fluorescence and resolution of electrophoretic bands. Preliminary evaluation of external conditions and recognition of the type of the substrate prior to casework bloodstain analysis is suggested.