In this study, the effect of light intermittence on solar disinfection of secondary treated wastewater was investigated. Synthetic secondary effluent was spiked with Escherichia coli and submitted to 3 different light intermittence regimes by circulating the effluent between a dark storage tank and three in-series illuminated reactors. The relative influence of the recirculation rate on bacterial inactivation was studied, in short (3–7 min) light regimes and a dark-to-light ratio of 2.04. Lower recirculation rates resulted in poorer disinfection results, showing the detrimental effect of longer dark storage periods on the removal efficiency. Also, longer time intervals were employed in batch tests, to investigate the effect of 1, 2 and 3-h dark intervals, during recreated solar disinfection conditions; fourteen different scenarios were tested. Three hours of continuous or cumulative illumination were proven enough to provide the necessary dose to damage bacteria irreparably, while interruption during these hours favored bacterial resistance. Finally, absence of regrowth was observed in all cases that derived from samples with null bacterial counts. However, when a fraction of viable bacteria was present at the end of the solar treatment, survival was favored.