Synchronizing Megachile rotundata (F.) nesting activity with alfalfa bloom is essential for ensuring optimal pollination for alfalfa seed production. This is achieved by timing the initiation of spring bee incubation so that adults will emerge ∼2 wk before peak bloom. If weather conditions change so as to delay the bloom, bee managers will commonly expose the developing bees to a period of low-temperature incubation to slow their development. We have previously demonstrated survival during low-temperature incubation can be significantly increased by using a fluctuating thermal regime (FTR) where the bees receive a daily pulse at 20°C. A FTR incubation protocol is composed of a number of different components, such as the base and pulse temperatures, and the duration and frequency of the pulse. In this investigation, the effect of the duration of the pulse (5–120 min) and the frequency of a pulse (twice daily to weekly) on the survival of developing M. rotundata was examined. A pulse as short as 5 min at 20°C increased survival of the developing bees as compared with the constant 6°C controls. Increasing the pulse duration induced a further increase in tolerance to 6°C. As with the pulse duration, increasing the pulse frequency from once weekly to twice daily had a significant effect on improving the bees tolerance to low-temperature incubation. This investigation further strengthens the argument that a FTR protocol is superior to using a constant low-temperature exposure for interrupting the spring incubation of M. rotundata.