Aims: The aim of this study was to apply the flow cytometry to Lactobacillus sakei strains, selected as potential autochthonous starters, to investigate dynamics and physiological heterogeneity of microbial behaviour under different stress conditions. Methods and Results: A simultaneous nucleic acid double-staining assay was applied to discriminate cell populations in different physiological states after exposure to heat (50 and 55 degrees C) and acid (pH 2.5 and 3.0) stresses. Alive cells with intact membranes, damaged cells still alive but with injured membranes, so with even a recovery ability, and dead cells with a permanent membrane damage were differentiated with a significant increase in damaged cells after stronger stress treatments. Conclusions: The existence and characteristics of subpopulations displaying heterogeneity in particular conditions are highly relevant, because specific subpopulations may show improved survival, changes and dynamics under stress conditions. Significance and Impact of Study: This assay has potential for physiological research on lactic acid bacteria and for application in the food industry. The assessment of intermediate physiological states in Lb.sakei strains with recovery possibility could be an important criterion for application of potential starter cultures. Application of flow cytometry and characterization of sorted subpopulations may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations.