Heterogeneity in the response of microbial cells to environmental conditions is inherent to every biological system and can be very relevant for food safety, potentially being as important as intrinsic and extrinsic factors. However, previous studies analyzing variability in the microbial response to thermal treatments were limited to data obtained under isothermal conditions, whereas in the reality, environmental conditions are dynamic. In this article we analyse both empirically and through mathematical modelling the variability in the microbial response to thermal treatments under isothermal and dynamic conditions.Heat resistance was studied for four strains of Listeria monocytogenes (Scott A, CECT 4031, CECT 4032 and 12MOB052), in three different matrices (buffered peptone water, pH 7 Mcllvaine buffer and semi-skimmed milk). Under isothermal conditions, between-strain and between-media variability had no impact in the heat resistance, whereas it was very relevant for dynamic conditions. Therefore, the differences observed under dynamic conditions can be attributed to the variability in the ability for developing stress acclimation. The highest acclimation was observed in strain CECT 4031 (10-fold increase of the D-value), while the lowest acclimation was observed in strain CECT 4032 (50% increase of the D-value). Concerning the different media, acclimation was higher in buffered peptone water and semi-skimmed milk than in Mcllvaine buffer of pH 7.0.To the knowledge of the authors, this is the first research work that specifically analyses the variability of microbial adaptation processes that take place under dynamic conditions. It highlights that microbial heat resistance under dynamic conditions are sometimes determined by mechanisms that cannot be observed when cells are treated in isothermal conditions (e.g. acclimation) and can also be affected by variability. Consequently, empirical evidence on variability gathered under isothermal conditions should be extrapolated with care for dynamic conditions.