The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperature (25°C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 - 95°C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45°C and 60°C). We have further compared the conformational impact of binding the efficiently transported substrate, alanine (Ala) to the much slower transported leucine (Leu). At elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25°C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. Furthermore, comparing the HDX signature of Ala vs. Leu we observe distinct differences that could correspond to the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of LeuT in the presence of Ala. Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.