Conformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of α-helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate-loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.