Characterizing pulsating flow in high-temperature, high-pressure engine exhaust gas is crucial for the development and optimization of exhaust energy recovery systems. However, the experimental investigation of engine exhaust pulses is challenging due to the difficulties in conducting crank angle-resolved measurements under these unsteady flow conditions. This study contributes to characterizing mass flow pulses from an isolated cylinder exhaust of a heavy-duty diesel engine using a single-pipe measurement system, developed for pulsating flow measurement. A Pitot tube-based approach is adopted to measure exhaust mass flow pulsations, complemented by fast temperature measurements obtained using customized unsheathed thin-wire thermocouples. The on-engine experiment is performed by isolating the in-cylinder trapped mass and the valve opening speed to produce different exhaust pulse waveforms. The adopted approach’s sensitivity in resolving instantaneous mass flows is evaluated analytically and experimentally, considering attenuated temperature measurement effects. Based on exhaust flow measurements, mass flow pulses are analyzed with regard to blow-down and scavenge phases. Under the load sweep, the main waveform change occurs during the blow-down phase, with pulse magnitude increasing with the load. In contrast, as the engine speeds up with a comparable trapped mass, the exhaust mass distribution in the blow-down phase decreases from 75.5% at 700 rpm to 41.9% at 1900 rpm. Additionally, it is observed that cycle-to-cycle variations in mass flow pulses align with combustion stability during the blow-down phase and are predominantly influenced by gas-exchange processes during the scavenge phase.