The variability of indicator bacteria over a fine resolution time scale on the order of minutes has yet to be fully understood. In this study, we collected more than 700 Escherichia coli samples at a 10- and 30-min resolution in an urban watershed in Houston. A Bacteria Diurnal Sag (BDS) marked with daytime exponential decay followed by an exponential nighttime regeneration was observed. This pattern was observed during all sampled events but varied depending on other variables. The concentrations during a 24-h period varied 1 to 5 orders of magnitude and the fecal load was at least 10 times lower than what would be obtained using a single morning E. coli measurement, the typical sampling scheme in most monitoring programs. Decay rates, ranging from 3.67 to 24.7/day, decreased E. coli concentrations to below the water-quality standards from 14:00 to 18:00 h and were strongly influenced by water temperatures and solar radiation intensities. Rapid regeneration occurred on the order of 9.41 to 64.1/day allowing E. coli concentrations to return to their pre-decay levels. The data indicated that four to six samples taken between 06:00 and 18:00 h may be sufficient to define the BDS depending on stream conditions, and that a threshold concentration of approximately 100 MPN/dl (most probable number in a deciliter) existed for the studied urban watershed. These findings have significant implications for water-quality monitoring, regulation, and compliance.