The effect of a 54°C heat exposure lasting 20 and 60 minutes on the invasiveness of 10 Listeria monocytogenes strains in the exponential and stationary phases was investigated. It was shown that heat exposure significantly reduced the L. monocytogenes count. A maximal reduction of 5 log took place after 60 minutes of heating. No significant differences in survival between exponential and stationary phases were observed for most strains. Twenty minutes of heat exposure resulted in a reduction of the invasiveness of L. monocytogenes, which was more pronounced in the exponential-phase bacteria. Prolongation of heating to 60 minutes was shown to have a significant impact on the invasiveness of five stationary-phase strains. It was demonstrated that heat exposure influences the survival of L. monocytogenes strains as expected. Also, the invasiveness was significantly changed in a time- and growth phase-dependent manner. Low reductions of bacterial counts in milder conditions, that is, 20 minutes of heat, were accompanied by a decrease of the invasiveness of all L. monocytogenes strains. Prolongation of heating time to 60 minutes resulted in significant reduction of bacterial numbers. However, bacteria, especially those in the stationary phase, which survived this treatment can become more invasive.