Background: Understanding how to enhance the motivation of inactive students to do physical activity is of great importance for school health. The purpose of the present study was to investigate the impact of an autonomous exercise training intervention on intrinsic motivation, physical activity intention, and health-related fitness of sedentary students in middle schools of Aliabad Katoul city, Golestan province, in 2019. Methods: The present causal-comparative field study was conducted on 45 middle school boys (mean age: 15.08 years) who were sedentary according to Godin-Shephard Questionnaire. The subjects were randomly assigned into three groups: Choice (autonomy training), No-Choice (training without autonomy), and control (no training). Subjects in Choice and No-Choice groups practiced physical fitness items (including flexibility and endurance) for eight weeks, such that the Choice group was allowed to the chosen order of exercises while the No-Choice group was trained in a predetermined order. Intrinsic motivation and physical activity intention were measured by questionnaire and health-related physical fitness components including flexibility, upper-body endurance, and cardiovascular endurance were evaluated by standard tests. One-way and mixed analysis of variance (ANOVA) were used to analyze the data. Tukey test was also used as follow-up test. Results: The participants in the Choice group reported higher intrinsic motivation (6.11±0.53) and physical activity intention (6.20±0.62) scores compared with other groups in post-test. Moreover, the results showed that autonomy training compared with training without autonomy and no training significantly increased intrinsic motivation (F=36.03, P<0.001) and physical activity intention (F=36.68, P<0.001). However, autonomy training did not improve physical fitness components such as flexibility (P=0.847), upper-body endurance (P=0.572), and cardiovascular endurance (P=0.982) more than non-autonomous training. Conclusions: These results may indicate that the feeling of autonomy during exercise training has a greater effect on psychological components (including intrinsic motivation and physical activity intention) compared with physical components (including physical fitness).