The paper presents a measurement campaign (electrical, thermal and user comfort) for the performance characterization of energy storage systems in real-world electric bicycles. Specific sensors were added to characterize three vehicles which differ for electric motor, energy storage system size and control strategies. The controller can implement energy recovery strategies when braking and change the level of electric assistance depending on the desired trade-off between the comfort of the driver and the battery duration. Experimental results show that a control strategy aiming at preserving the SOC (State-Of-Charge), together with regenerative braking, can ensure very long battery duration with no need of recharge. The SOC is kept at about 50% for a long period. Instead, control strategies optimizing the full comfort of the driver by maximizing the level of assistance can ensure real-world e-bicycle missions of about 2 h and 40 km, when the SOC of the battery drops down from 95% to 5%.