The UP segment is the normal isoelectric reference level for ECG wave amplitude measurements but becomes obscured at high heart rates. The aim was to identify alternative reference levels suitable for use across a wide range of heart rates. 12-lead ECGs were recorded from 10 healthy subjects before and immediately following exercise. Amplitudes of the UP segment, Q wave, end of T wave (Tend) and zero voltage level, all relative to PQ level were measured from V3. The performances of beat waveform averaging (AvgBeat) and mathematical averaging of separate beat measurements (AvgVal) on reducing the influence of noise and measurement errors were compared. Due to merging of P and U waves at high heart rates, the UP segment amplitude was measureable in only approximately 71% when the heart rate was over 120 b/min. Both the UP segment amplitude and Tend amplitude tended to be overestimated at high heart rates. The standard deviations (SDs) were 0.02, 0.005, 0.021 and 0.016 mV for UP segment, Q wave, end of T wave (Tend) and zero voltage levels over the range of heart rates when using AvgVal. The SDs of amplitudes measured by AvgBeat and AvgVal methods were significantly lower than those measured from single beats (p < 0.05) for almost all features. Generally, these two methods achieved comparable performance on reducing measurement variability.