Because of their characteristics, which have been continuously improved during the last years, Lithium ion batteries were proposed as an alternative viable solution to present fast-reacting conventional generating units to deliver the primary frequency regulation service. However, even though there are worldwide demonstration projects where energy storage systems based on Lithium-ion batteries are evaluated for such applications, the field experience is still very limited. In consequence, at present there are no very clear requirements on how the Lithium-ion battery energy storage systems should be operated while providing frequency regulation service and how the system has to re-establish its SOC once the frequency event has passed. Therefore, this paper aims to investigate the effect on the lifetime of the Lithium-ion batteries energy storage system of various strategies for re-establishing the batteries’ SOC after the primary frequency regulation is successfully delivered.