Background: Left ventricular ejection fraction (LVEF) estimation using adenosine stress myocardial perfusion imaging (MPI) can be challenging. The short half-life of adenosine and the guideline-recommended adenosine infusion stop during Rubidium-82 acquisition protocol may affect the accuracy and repeatability of the LVEF measures. Methods: This study comprised 25 healthy volunteers (median age 23 years) who underwent repeat myocardial perfusion imaging (MPI) sessions employing Rubidium-82 PET/CT. A guideline-recommended reconstruction protocol was used for both rest and adenosine stress MPI (150-360 s post-radiotracer injection, standard_recon). For the stress MPI protocol, two additional reconstruction protocols were considered; one was employing 60 seconds data (150-210 seconds, short_fixed) and the other a dynamic frame window based on the bolus arrival of Rubidium-82 in the heart until 210 seconds (x-210 seconds, short_individual). We report rest and stress LVEF, the LVEF reserve, and the LVEF reserve repeatability. Results: Differences in the LVEF assessments were observed between the guideline recommended and alternative reconstruction protocol (LVEF stress MPI: standard_recon = 68 ± 7%, short_fixed = 71 ± 7% (P =.08), short_individual = 72 ± 7% (P =.04)), and the LVEF reserve was reduced for the guideline-recommended protocol (standard_recon = 7.8 ± 3.5, short_fixed = 10.1 ± 3.7, short_individual = 10.5 ± 3.6, all P <.001). The best repeatability measures were obtained for the short_individual protocol (repeatability: standard_recon = 45.3%, short_fixed = 41.2%, short_individual = 31.7%). Conclusion: We recommend using the short_individual reconstruction protocol for improved LVEF repeatability and reserve assessment. Alternatively, in centers with limited technical support we recommend the use of the short_fixed protocol.