Non-nutritive sweeteners (NNSs) elicit a multitude of endocrine effects in vitro, in animal models, and in humans. The best-characterized consequences of NNS exposure are metabolic changes, which may be mediated by activation of sweet taste receptors in oral and extraoral tissues (e.g., intestine, pancreatic β cells, and brain), and alterations of the gut microbiome. These mechanisms are likely synergistic and may differ across species and chemically distinct NNSs. However, the extent to which these hormonal effects are clinically relevant in the context of human consumption is unclear. Further investigation following prolonged exposure is required to better understand the role of NNSs in human health, with careful consideration of genetic, dietary, anthropometric, and other interindividual differences.