We used single-pair fluorescence resonance energy transfer (spFRET) to track distance changes between domains of fluorescently labeled calmodulin (CaM) on the sub-millisecond time scale. In most cases, CaM remained in the same conformational substate over time periods of up to 1 ms, showing that conformational interchange occurs on a longer time scale. However, in some instances, apparent transitions between conformational substates could be detected. The magnitude of sub-millisecond motion within the dominant conformational substate also revealed fluctuations in distance between domains that were dependent on pH and ionic strength.