Abstract The broad goal of this project is to determine whether shifting the temporal distribution of macronutrient intake impacts metabolic markers of health, which holds great translational value for vulnerable populations, including night shift workers. Night work is increasingly prevalent and a risk factor for type 2 diabetes (T2D). We and others have shown that circadian misalignment itself, i.e., the misalignment between the eating/fasting cycle and the central circadian timing system, leads to impaired glucose tolerance and decreased insulin sensitivity, even in chronic shift workers. Circadian misalignment is thus a likely mechanism contributing to the increased T2D risk in night workers. Because night work is not likely to go away anytime soon, countermeasures against these adverse metabolic effects are desperately needed. Our preliminary data from stringently-controlled circadian experiments suggests that glucose tolerance, beta-cell function, and diet-induced thermogenesis are increased in the circadian morning compared to the circadian evening, and that—in contrast—fat tolerance is increased in the circadian evening compared to the circadian morning. Based on these insights, we have developed a novel and translational approach that combines the concepts of the importance of WHAT we eat with the importance of WHEN we eat: scheduling high-carb intake for the circadian morning and high-fat intake for the circadian evening (C/F; expected to be favorable) as compared to vice versa (F/C, high-fat for circadian morning and high-carb for circadian evening), without changing 24-h caloric or macronutrient intake. Using two highly-controlled, within-subject, randomized, crossover protocols (one under circadian alignment, one under circadian misalignment), we will test the hypotheses that high-carb intake during the biological morning and high-fat intake during the biological evening (C-F) compared to vice versa (F-C) leads to: higher glucose tolerance (Aim 1); higher diet-induced thermogenesis (Aim 2); and higher fat tolerance (Exploratory Aim 3). We will test these aims in a robust and sophisticated study design: (1) without disturbing sleep during the day; (2) without requiring extended fasting during wakefulness at night; (3) without changing the caloric content per meal; and (4) without changing 24-h caloric or 24-h macronutrient intake. Knowledge on the health impacts of macronutrient intake timing is not only important for shift workers but also for the general population. Therefore, these questions will not only be addressed under circadian misalignment but also circadian alignment.