Forest trail difficulty levels must be investigated to promote their safe usage. However, exercise-based physiological changes are difficult to estimate in a forest environment. Hence, studies comparing indoor and outdoor environmental activities are required. We aimed to investigate the physiological traits associated with different usage grades of national forest trails in the Republic of Korea. We conducted both field and indoor experiments to measure different variables of 20 healthy participants aged 40–50 years to evaluate the exercise intensity and physiological effects of hiking on these trails. Significant variations in physiological responses were observed based on trail difficulty, with the highest heart rate and energy expenditure recorded during the most challenging uphill segments. Specifically, the heart rate increased from 115.4 ± 13.7 bpm in easy segments to 140.3 ± 15.6 bpm in difficult segments, whereas energy expenditure ranged from 404.1 ± 112.2 kcal/h to 518.1 ± 131.0 kcal/h. This study highlights the importance of considering both objective trail difficulty and users’ physiological reactions, including perceived exertion, to enhance user safety and optimize the health benefits of national forest trails. These findings provide essential data for developing comprehensive exercise programs and improving the national forest trail usage grade system.