Background: One of the resistance exercise variables is the sequence of activities (1). Newsholme and colleagues (1978) introduced the hypothesis that prolonged exercise due to central fatigue, increased serotonin, and reduces the performance (2). .Furthermore, cortisol by reducing tryptophan could affect on serotonin (3). Methods: 15 students from Kurdish university were selected and randomized into two groups. Movements were done with a combination of upper and lower body activity, and took the large and small muscles. In a cross-sectional design, half of the subjects followed protocol A (from large to small muscle), and the others protocol B (from small to large). One minute rest between each exercise was set and rest intervals between each move were two minutes. Results: Serotonin concentration in B protocol immediately after exercise than before exercise significantly increased (p= 0/001), but no change was observed in A protocol. Moreover, changes in cortisol in both protocols, revealed non- significant decrease immediately after exercise. Also, in B protocol, rate of perceived exertion was more of A protocol. Discussion and Conclusion: The research results can be said about the changes in serotonin, A protocol is a protocol that provides greater fatigue. This increase was consistent with the more rate of perceived exertion in the B protocol. Aftermore, a possible mechanism of serotonin -induced changes is not cortisol, which is consistent with some previous findings (3).