The aim of this study was to determine the effect of the cupping therapy on the muscles and autonomic nervous system. Five healthy participants were recruited for the arm cranking tests. The protocols include a 1st 12-min bout, 20-min recovery, and 2nd 12-min bout. The cupping therapy (negative pressure of 300–450 mmHg on the upper back and shoulder muscles) was applied during the recovery period of experimental trials. The median frequency (MDF) of electromyographic signals and the heart rate variability (HRV) were used to objectively quantify the degree of muscle fatigue and cardiovascular regulations. The repeated measures ANOVA was applied to determine the differences. Our results showed that MDF was significantly higher in the experimental trials but the HRV was not significantly different between the experimental and control trials. Our study indicates that the role of cupping therapy may reduce muscle fatigue but do not alter the cardiorespiratory controls.