Compression heating characteristics of different pressure transmitting fluids [three different concentrations (75:25, 50:50, 25:75) of water–glycol mix and sodium benzoate (2%) solutions] and their influence on inactivation of spores of Bacillus subtilis in phosphate buffer (0.067 M, pH 7.0) during high pressure processing (HPP) were studied. Experiments were conducted using a pilot scale food processor. Pressure transmitting fluids containing highest percentage of glycol (25:75 water–glycol mix) showed highest temperature increase while 2% sodium benzoate solution showed least temperature increase during high pressure processing. The target pressure, holding time, compressibility, initial temperature, and the rate of heat loss to the surroundings primarily influenced the apparent temperature increase of pressure transmitting fluid in a vessel during HPP. The temperature change was further influenced by the fluid properties such as viscosity, specific heat and thermal conductivity. Use of sodium benzoate solution as pressure-transmitting fluid resulted in highest inactivation of B. subtilis spores. Change in pressure transmitting fluid temperature as a result of compression heating and subsequent heat transfer should be considered in inactivation of bacterial spores by HPP.