A rest–pause (RP) technique involves performing one or more repetitions at high resistance to failure, followed by a short rest before performing one or more repetitions. These techniques can affect neuromuscular conditions and fatigue by changing the rest time between repetitions. This study compared the effect of 12 weeks of RP and traditional resistance training (TRT) on myokines (myostatin (MSTN), follistatin (FLST) and insulin-like growth factor-1 (IGF-1)) and functional adaptations. The study recruited 29 men between the ages of 20 and 30 who had performed resistance training for at least 6 to 12 months. Participants were randomly divided into three groups: RP, TRT, and control; resistance training was performed 3 days per week for 12 weeks. The training methods of the two groups were largely similar. The results showed that RP increased IGF-1 and FLST/MSTN more than the TRT group (% change = 19.04, % change = 37.71), and only the RP and TRT groups had significant changes in the FLST/MSTN ratio compared to the control group (p < 0.001 and p = 0.02, respectively). In addition, FLST levels increased and MSTN decreased in the RP and TRT groups, but the rate of change in FLST was significant in the RP and TRT groups compared to the control group (p = 0.002 and p = 0.001, respectively). Leg press and bench press strength, and arm and thigh muscular cross-sectional area (MCSA) increased more in the RP group than in the others, and the percentage of body fat (PBF) decreased significantly. The change between strength and MCSA was significant (p ≤ 0.05), and the PBF change in RP and TRT compared to the control (ES RP group = 0.43; ES TRT group = 0.55; control group ES = 0.09) was significant (p = 0.005, p = 0.01; respectively). Based on the results, the RP training technique significantly affects strength and muscle hypertrophy more than the TRT method, which can be included in the training system to increase strength and hypertrophy.