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Purpose: To define the optimal cardiac short-axis cine acquisition protocol for the assessment of the left and rightventricular functions. Materials and methods: 20 volunteers were recruited and breath-hold CINE images were acquired on a Siemens Prisma 3T MRI. Four short-axis acquisition planes were defined from the 4-chamber view. AV Junctions: short-axis slices parallel to the plane that cuts through the external right and left atrioventricular junctions. Left AV Junctions: short-axis slices parallel to the plane that cuts through both left atrioventricular junctions. Septum: short-axis slices perpendicular to the septum with one cutting through the septum junction. LongAxis: short-axis slices perpendicular to the long axis with one cutting through the septum junction. Intra and inter reproducibility was assessed using Bland-Altman coefficient of variation (CV) and Lin’s concordance correlation coefficient (CCC). The influence of the protocol on the ejection fraction (EF) and stroke volume (SV) was quantified statistically using pair-wise CV and Pearson’s correlation coefficient R2. Results: All protocols led to high reproducibility for the LV EF (mean intra CV = 3.83%, mean inter CV = 4.81%, lowest CV = 4.20% (AV junctions) and highest CV = 5.24% (Left AV Junctions)). Reproducibility of the RV measurements was lower (mean intra CV = 7.84%, mean inter CV = 9.17%). Septum protocol led to significantly lower variability compared to the other 3 protocols for RV EF (CV = 7.62% (Septum), CV = 8.42% (Long Axis), CV = 9.54% (Left AV Junctions) and CV = 11.08% (AV Junctions) with Lin’s CCC varying from 0.4 (AV Junctions) to 0.69 (Septum) for inter-observer reproducibility). No differences in group average for clinical parameters was found for both LV and RV clinical measurements. However, patient-specific RV EF evaluation is dependent on the chosen protocol (CV = 9.95%, R2 = 0.52). Conclusion: Based on the results of the study cine mode short-axis acquisitions should be planned perpendicular to the septum in order to guarantee optimal RV and LV measurements. Keywords: Cine MRI, Short-axis acquisition, Reproducibility study, Right ventricle, Left ventricle