Macronutrient substitutes (MNS) are food ingredients designed to replace the organoleptic and/or functional properties of macronutrients such as fats or sugars in processed foods. Because they may be consumed in large quantities daily, traditional methods of safety evaluation are inappropriate. Conventional safety factors cannot be used in extrapolating animal data to humans due to the limitations of administering very large doses of MNS to animals. The proper evaluation of the safety of MNS involves appropriate studies in animals and humans including comparative biodispositional studies, genotoxicity and cytotoxicity studies, reproductive and developmental studies, mechanistic studies, digestive and fermentation studies, nutritional studies, and studies involving humans with special focus on gastrointestinal function. Guidelines for the proper conduct of human studies were presented and these include the use of competent investigators and IRB-approved protocols and the use of adequate numbers of healthy male and female volunteers. Postmarketing surveillance is the final step in the safety evaluation process for macronutrient substitutes. It was concluded that MNS should be evaluated on a case-by-case basis.