The extended endocannabinoid system, also termed endocannabinoidome, participates in multiple metabolic functions in health and disease. Physical activity can both have an acute and chronic impact on endocannabinoid mediators, as does diet. In this crossover randomized controlled study, we investigated the influence of diet on the peripheral response to acute maximal aerobic exercise in a sample of active adult women (n = 7) with no underlying metabolic conditions. We compared the impact of 7-day standardized Mediterranean diet (MedDiet) and control diet inspired by Canadian macronutrient intake (CanDiet) on endocannabinoidome and short-chain fatty acid metabolites post maximal aerobic exercise. Overall, plasmatic endocannabinoids, their congeners and some polyunsaturated fatty acids increased significantly post maximal aerobic exercise upon cessation of exercise and recovered their initial values within 1 h after exercise. Most N-acylethanolamines and polyunsaturated fatty acids increased directly after exercise when the participants had consumed the MedDiet, but not when they had consumed the CanDiet. This impact was different for monoacylglycerol endocannabinoid congeners, which in most cases reacted similarly to acute exercise while on the MedDiet or the CanDiet. Fecal microbiota was only minimally affected by the diet in this cohort. This study demonstrates that endocannabinoidome mediators respond to acute maximal aerobic exercise in a way that is dependent on the diet consumed in the week prior to exercise.