Rapid and direct viability assessment of Escherichia coli in filtered, sterile lake water was possible using multiparameter flow cytometry. Fluorescent dyes were used as probes for different cellular functions (membrane potential, membrane integrity and intracellular enzyme activity), which were correlated with the ability of the cells to respond to nutrient addition while in a stressed state. Measurement of several criteria circumvented limitations imposed by other methods, and provided extensive evidence for the validity of the methods for monitoring cell viability during adoption of a viable‐but‐non‐culturable state in starved E, coli. Macromolecular staining was concomitantly used to monitor changes in cellular protein, RNA and DNA as additional indicators of physiological status during starvation/stress.