Purpose The main objective of this study was to investigate the effect of time of contraction and rest on the masseter and temporal muscles activity. Methods 49 female subjects between 18 and 30 years of age were divided into TMD (n: 26) and control groups (n: 23). Surface electromyograph was used to evaluate the anterior temporal and masseter muscles during contraction and rest protocols. The root means square, median frequency and slope coefficient of the linear regression line parameters were analyzed. Results A significant effect of time in the contraction and rest muscle protocols was found. TMD patients showed a significant decrease in median frequency in the right masseter muscle and the slope coefficient in the right temporal muscle during the contraction protocol to control subjects. Conclusion Despite the TMD patients presented with higher fatigue susceptibility compared to the control group, both groups must meet the maximum time of 5 s of maximum voluntary contraction and at least 30 s rest between successive contractions of masticatory muscles during clinical or research assessment protocols.