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Abstract Background Different dietary protein sources are supposed to have various effects on metabolic responses and arterial stiffness in the postprandial period. This study aims to assess the postprandial effects of dietary protein sources, including animal-based protein (AP) and plant-based protein (PP), as part of a high-protein breakfast on appetite response, energy metabolism, and arterial stiffness in overweight and obese men. Methods This acute randomized crossover clinical trial will be conducted at the Persian study research center at Imam Reza Hospital, affiliated with the Mashhad University of Medical Sciences, located in the northeast of Iran. Forty-six healthy overweight, and obese men aged 18–60 years will be enrolled based on the eligibility criteria. The subjects will complete two interventions (high-protein AP and PP meals) with 1 week washout period. The primary outcome will be the acute effect of the two test meals on appetite response, energy metabolism parameters, including resting metabolism rate (RMR), diet-induced thermogenesis (DIT), and substrate oxidation (SO), and arterial stiffness indices, including pulse wave velocity (PWV) and pulse wave analysis (PWA). The secondary outcomes include changes in lipemia, glycemia, and insulinemia. Discussion The findings of this study will provide novel insight regarding the acute effects of different protein sources on energy metabolism, appetite, and arterial stiffness as a significant cardiovascular disease (CVD) risk factor. It will help dieticians develop effective and efficient meal plans to improve weight reduction and maintenance in overweight/obese individuals. Trial registration Iranian Registry of Clinical Trials; code: IRCT20211230053570N1; registered on February 10, 2022