Heart rate asymmetry (HRA) could capture valuable dynamical properties from the electrocardiographic RR interval time-series that are helpful for evaluating the cardiovascular functioning. Several metrics derived from the Poincaré plot have been established for assessing HRA such as the slope index (SI) and the area index (AI). In the current study, we aimed to examine how different reference points and reference lines affect the calculations of SI and AI. To understand their performance, two case studies that were to classify subjects with (1) arrhythmias and (2) congestive heart failure, respectively, from normal controls were performed. To examine whether these effects depend on data lengths, the case studies were performed on both long-term heartbeat interval time-series and short-term segments. Our results showed that different reference points or reference lines could strongly affect the performance of both SI and AI, especially when short-term data were being analyzed. Using the minimum of data as the reference point might be a conservative solution in application but a spectrum of SI or AI measurements with multiple reference points and reference lines are highly recommended.