There is considerable interest in whether non-nutritive sweeteners are sensed in the gastrointestinal tract to modulate appetitive or absorptive responses to ingested carbohydrate. We determined the effect of a panel of non-nutritive sweeteners, aspartame, saccharin and acesulfame-K, delivered in doses that would be consumed in normal usage. Each was given in combination with glucose, assessing their effect on glycemic responses and appetite in 10 healthy human subjects. There was no additional effect of aspartame or saccharin on the blood glucose response to oral glucose at any time point, although acesulfame-K exerted a small effect. However, none had an effect on perceptions of hunger or fullness. We conclude that there is no consistent evidence that non-nutrient sweeteners, when acutely consumed with glucose in dietetically relevant doses, have a class effect in modulating blood glucose in healthy human subjects. However, acesulfame-K may require further exploration.