Both nutrition and exercise are known to affect metabolic regulation in humans. Sirtuins are essential regulators of cellular energy metabolism; SIRT1, SIRT3, and SIRT4 have a direct effect on glycolysis, oxidative phosphorylation, and fatty acid oxidation. This cross-sectional study investigates the effect of different diets on exercise-induced regulation of sirtuins. SIRT1 and SIRT3–SIRT5 were measured in blood from omnivorous, lacto-ovo vegetarian, and vegan recreational runners (21–25 subjects, respectively) before and after exercise at the transcript, protein, and enzymatic levels. SIRT1, SIRT3, and SIRT5 enzyme activities increased during exercise in omnivores and lacto-ovo vegetarians, commensurate with increased energy demand. However, activities decreased in vegans. Malondialdehyde as a surrogate marker of oxidative stress inversely correlated with sirtuin activities and was elevated in vegans after exercise compared to both other groups. A significant negative correlation of all sirtuins with the intake of the antioxidative substances, ascorbate and tocopherol, was found. In vegan participants, increased oxidative stress despite higher amounts of the antioxidative substances in the diet was observed after exercise.