Caloric restriction (CR) induces weight loss, but is associated with rapid weight regain upon return to ad libitum feeding. Our aim was to investigate effects of the macronutrient composition of the diet on weight loss and regain in elderly mice. Males, 18 months old, of the C57BL/6J strain were subjected to 4-week 30% CR followed by 4 weeks of ad libitum refeeding on either high-carb (HC), high-fat (HF) or high-protein (HP) diets (n = 22 each). Mice (n = 11) fed a chow diet ad libitum served as a control group (CON). Body mass and food intake were monitored daily. Twenty-four-hour indirect calorimetry was used to assess energy expenditure and substrate oxidation. Muscle and fat mass were evaluated with dissection of the tissues. Serum leptin and ghrelin levels were also measured. CR-induced weight loss did not differ between the diets. Weight regain was particularly fast for HF as mice overshot their initial weight by 12.8 ± 5.7% after 4-week refeeding when HC and HP mice reached the weight of the CON group. Weight regain strongly correlated with energy intake across the groups. The respiratory exchange ratio was lower in HF mice (0.81 ± 0.03) compared to HC (0.94 ± 0.06, p &lt; 0.001), HP (0.89 ± 0.04, p &lt; 0.001) and CON mice (0.91 ± 0.06, p &lt; 0.01) during the refeeding. Serum leptin levels were higher in HF mice (1.03 ± 0.50 ng/mL) compared to HC (0.46 ± 0.14, p &lt; 0.001), HP (0.63 ± 0.28, p &lt; 0.05) or CON mice (0.41 ± 0.14, p &lt; 0.001). Thus, CR induces similar weight loss in aging mice irrespective of the diet’s macronutrient composition. An HF diet leads to excessive energy intake and pronounced gain in body fat in spite of increased fat oxidation and serum leptin during the refeeding after CR. 2023 https://creativecommons.org/licenses/by/4.0/ Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). Caloric restriction (CR) induces weight loss, but is associated with rapid weight regain upon return to ad libitum feeding. Our aim was to investigate effects of the macronutrient composition of the diet on weight loss and regain in elderly mice. Effects of dietary composition on this weight rebound remain poorly understood. Studies of C57BL/6 mice are well suited to address this lack of knowledge as these mice are prone to obesity and show similar metabolic dysfunctions as humans [ 13 ]. Along with increased life expectancy and food availability, obesity is becoming increasingly prevalent in the elderly [ 14 ]. The aim of our current study was to investigate effects of dietary macronutrient composition on physiological adaptations of old C57BL/6 mice to 30% CR followed by a period of ad libitum feeding. This was performed by subtracting the weight of food leftovers at the end of each week from the initially provided food weight with corrections for a humidity effect on the pellets’ weight. 2.2. Caloric Restriction and Refeeding Initially, 18-month-old C57BL/6J mice were subjected to 30% CR for 4 weeks on (1) high-fat (HF; 60%, 20% and 20% kcal from fat, carbohydrate and protein, respectively), (2) high-carbohydrate (HC; 20%, 60%, 20% kcal from fat, carbohydrate and protein, respectively) or (3) high-protein (HP; 30%, 35% and 35% kcal for fat, carbohydrate and protein, respectively) diets. During the entire refeeding period, the HF group consumed 87 ± 14% more food energy than during the previous period of caloric restriction of the same duration, and this increase was significantly greater compared to the other diet groups (HC and HP increases were 57 ± 11% and 55 ± 12%, respectively; p < 0.001). Association between energy intake and weight gain during the refeeding is shown in Figure 1 d. There was a strong positive correlation between changes in energy intake and body mass (r = 0.95, p < 0.001). The CON group maintained a consistent energy intake throughout the study and their body mass remained relatively stable. Our results show that these effects mostly depend on energy status. There were no significant differences between the diets applied in body weight or tissue loss during CR. However, the fastest weight and fat regain was observed in mice refed using a high-fat diet. This was observed in spite of elevated fat oxidation in these mice, as weight regain correlated with energy intake. Our results suggest that weight and fat gain is primarily dependent on the energy intake, which is linked to energy density of the food rather than ability to oxidize fat during refeeding. In addition to leptin and ghrelin data, it would also have been useful to examine the levels of certain gut-derived hormone incretins (e.g., GIP, GLP-1) related to glucose metabolism and mediating satiety [ 37 ]. 5. Conclusions Our findings highlight that the effects of macronutrient allocation in the diet on body composition in aged mice are dependent on energy balance. During isocaloric energy restriction, the dietary macronutrient composition has a relatively smaller impact on weight and fat loss than caloric restriction itself. However, when mice are exposed to ad libitum refeeding, the high-fat and low-carbohydrate diet leads to the least favorable body composition outcomes, which induce rapid weight regain that overshoots initial weight levels before caloric restriction, despite increased fat oxidation and high serum leptin levels associated with this diet. Increase in body and fat mass with this diet is explained with increased ad libitum energy intake. Figure 2 Weights of dissected mouse tissues and organs, including hindlimb muscles ( a – c ), fats from different sampling sites ( d – f ), liver ( g , h ) and heart ( i , j ), after a 4-week caloric restriction (CR) followed by 4-week post weight loss ad libitum refeeding (P-CR) with diets of different macronutrient composition (high-carbohydrate, HC; high-fat, HF;