Background/Objectives: Nutrient profiling of foods is defined as the science of ranking or classifying foods based on their nutrient content. Nutrient profiles can be calculated based on 100 g or 100 kcal of food or on standard serving sizes. The objective of this study was to compare the performance of nutrient profiles based on 100 g, 100 kcal and government-mandated serving sizes, and to identify the optimal base of calculation. Subjects/Methods: Nutrient profiles tested were composed of positive subscores based on nutrients to encourage and negative subscores based on nutrients to limit. Alternative profiles, computed using different bases of calculation, were used to rank order 378 commonly consumed foods from a food frequency instrument. Profile performance was tested with respect to the foods’ energy density. Results: Serving sizes, defined by the US Food and Drug Administration as reference amounts customarily consumed (RACC), were inversely linked to energy density of foods. Positive subscores based on 100 kcal were equivalent to those calculated using RACC values. Negative subscores performed better when based on 100 g as opposed to 100 kcal. Conclusions: Models based on serving sizes and on 100 kcal were preferable for positive subscores and models based on 100 g of food were preferable for negative subscores. RACC-based profiles may represent an attractive option for the US consumer