Evidence linking sugar-sweetened beverage (SSB) consumption to weight gain and other negative health outcomes has prompted many individuals to resort to artificial, non-nutritive sugar (NNS) substitutes as a means of reducing SSB intake. However, there is a great deal of controversy regarding the biological consequences of NNS use, with accumulating evidence suggesting that NNS consumption may influence feeding and metabolism via a variety of peripheral and central mechanisms. Here we argue that NNSs are not physiologically inert compounds and consider the potential biological mechanisms by which NNS consumption may impact energy balance and metabolic function, including actions on oral and extra-oral sweet taste receptors, and effects on metabolic hormone secretion, cognitive processes (e.g. reward learning, memory, and taste perception), and gut microbiota.