Fasting animals are believed to sequentially switch from utilizing one metabolic substrate to another in the order of carbohydrates, to lipids, then to proteins. The timing of these physiological transitions are traditionally estimated using indirect measures of substrate oxidation including, changes in RER, blood metabolites, or respiratory enzyme activities. Here we show that the oxidation of distinct nutrient pools in the body (lipids, proteins, and carbohydrates) that are gradually enriched in 13C can be directly quantified using 13CO2-breath testing. Seventy-two Swiss Webster mice were raised to adulthood on diets supplemented with 13C-1-L-leucine, 13C-1-palmitic acid, 13C-1-D-glucose, or no tracer. Mice were then fasted for 72 hours during which VO2, VCO2, δ13CO2, body temperature (Tb), and blood metabolites (i.e., glucose, ketone bodies, and triacylglycerides) were continually measured. The fasting mice exhibited reductions in Tb (4.6°C), body mass (29%), BMR (24%), and RER (0.18), and significant changes in blood metabolites, but these responses were not useful to clearly identify specific changes in fuel mixture. Direct measurements of endogenous nutrient oxidation by way of 13CO2-breath testing revealed a rapid crash in the rate of oxidation of carbohydrates from 47% to 27% of the total energy expenditure during the first 6 hours without food. This response was mirrored by a coincidental increase in rate of endogenous lipid oxidation from 31% to 52%. A final transient peak in carbohydrate oxidation occurred between 8-18 hours indicating the transition between Phase I and II. A period of protein sparing between 8-12 hours was observed where endogenous protein oxidation accounted for as little as 9% of the total energy expenditure. Thereafter, protein oxidation continually increased accounting for as much as 25% of the total energy expenditure by 72 hours, suggesting that these mice exhibit a gradual, not an abrupt, transition from Phase II to III. This study demonstrates that direct measurements of substrate oxidation address a gap in our understanding of the timing and magnitude of sequential changes in substrate oxidation that occur during prolonged fasting and starvation.