Thermal characterization of lithium-ion batteries is essential to improve an efficient thermal management system for lithium-ion batteries. Besides, it is needed for safe and optimum application. The investigated lithium-ion battery in the present research is a commercially available lithium titanate oxide-based lithium-ion battery, which can be used in different applications. Different experimental facilities were used to measure lithium-ion battery heat generation at different operating conditions and charge and discharge rates in this investigation. Isothermal battery calorimeter is the exclusive calorimeter globally, suitable for lithium-ion batteries’ accurate thermal measurements. Pulse charge and discharge in different increments of state of charge were applied to the lithium titanate oxide-based lithium-ion battery to designate the heat generation of the lithium-ion battery cell. Three different cases were studied. The precise effects of different state-of-charge levels and current-rates on lithium-ion battery total generated heat was investigated. The maximum heat generation during 13 A, 40 A, 50 A, 60 A and 100 A pulse discharges were 0.231 Wh, 0.77 Wh, 0.507 Wh, 0.590 Wh and 1.13 Wh correspondingly. It could be inferred that in the case of periodic charge and discharge pulses applied to the lithium titanate oxide-based lithium-ion battery, important parameters including state of charge, current rates, initial cycling, and temperature have a significant influence on total generated heat.