Abstract Context Endocannabinoids are suggested to play a role in energy balance regulation. Objective We aimed to investigate associations of endocannabinoid concentrations during the day with energy balance and adiposity and interactions with 2 diets differing in protein content in participants in the postobese phase with prediabetes. Design and Participants Participants (n = 38) were individually fed in energy balance with a medium protein (MP: 15:55:30% of energy from protein:carbohydrate:fat) or high-protein diet (HP: 25:45:30% energy from P:C:F) for 48 hours in a respiration chamber. Main Outcome Measures Associations between energy balance, energy expenditure, respiratory quotient, and endocannabinoid concentrations during the day were assessed. Results Plasma-concentrations of anandamide (AEA), oleoylethanolamide (OEA), palmitoyethanolamide (PEA), and pregnenolone (PREG) significantly decreased during the day. This decrease was inversely related to body mass index (AEA) or body fat (%) (PEA; OEA). The lowest RQ value, before lunch, was inversely associated with concentrations of AEA and PEA before lunch. Area under the curve (AUC) of concentrations of AEA, 2-AG, PEA, and OEA were positively related to body fat% (P < .05). The HP and MP groups showed no differences in concentrations of AEA, OEA, PEA, and PREG, but the AUC of 2-arachidonoylglycerol (2-AG) was significantly higher in the HP vs the MP group. Conclusions In energy balance, only the endocannabinoid 2-AG changed in relation to protein level of the diet, whereas the endocannabinoid AEA and endocannabinoid-related compounds OEA and PEA reflected the gradual energy intake matching energy expenditure during the day.