This study aimed to gain more in-depth knowledge of the mechanisms involved in microbial inactivation by pulsed electric fields (PEF) to understand the tailing observed in survival curves of Salmonella Typhimurium (STCC 878). The comparison of the inactivation achieved by the application of one train of pulses with those obtained with pulses applied in two trains shows that the tail of the survival curves was a consequence of a transient increment of the microbial resistance to the effect of the electric field in a proportion of the cells. After some time following the application of the first pulse train, cells became again sensitive to the second train, and tailing tended to disappear. The required time was highly dependent on the characteristics of the incubation medium. Similar effects were observed when the treatments were validated on whole milk and orange juice. This study has demonstrated by the first time on microbial cells the benefits of splitting the delivered PEF treatment in two trains with a period of delay between them. Therefore, this insight opens up the possibility of developing new strategies to achieve the required inactivation levels to guarantee food safety by moderate PEF treatments.