The adhesive force between two contact surfaces often leads to an increase in the friction force of the rubber seal O-ring after a certain dwell time, forming dwell time effects and affecting the reliability of sealing. The dwell time effect may result in substantial instability with respect to the frictional behavior of rubber O-rings, which needs to be carefully taken into account in the design of rubber seals. Therefore, in this paper, the dwell time effect of the friction force was studied experimentally for intermittent reciprocating rubber seal O-rings coupled with stainless steel 316L and a sealing air medium. The friction force of three kinds of rubber materials, including fluorine rubber (FPM), silicone rubber (SI), and nitrile rubber (NBR), was measured under different dwell times, compression ratios, and seal pressure. The results showed that there was a rolling frictional force, and the second peak value of the frictional force caused by the O-ring's rolling under shear action and after the maximum static frictional force was observed at the starting stage of reciprocating motion. For FPM O-rings, the rolling friction force was much greater than the maximum static frictional force at about four times the value of the compression ratio at 9% and seal pressure at 0; moreover, the force was much greater at greater compression ratios. The dwell time effect was significant in the friction forces of rubber O-rings. The friction force increases with an increase in dwell time. The increase in maximum static friction force exceeded 50% after 5 dwell days. The increase in seal pressure led to the disappearance of the rolling friction feature and the rapid increase in friction during the starting stage. Under gas seal pressure conditions, the dwell time effect still led to a significant increase in friction force. The obtained results might provide guidance for the material selection of sealing designs.