The aim of the current study was to assess the scan–rescan reproducibility of left ventricular (LV) kinetic energy (KE), viscous energy loss (EL) and vorticity during diastole from four-dimensional flow magnetic resonance imaging (4D flow MRI) in healthy subjects. Twelve volunteers (age 27 ± 3 years) underwent whole-heart 4D flow MRI twice in one session. In-scan consistency was evaluated by correlation between KE and EL. ELindex was computed to measure the amount of EL relative to KE over diastole. Scan–rescan analysis was performed to test reproducibility of volumetric measurements of KE, EL, ELindex and vorticity in the LV over early (E) and late (A) diastolic filling. In-scan consistency between KE and EL was strong-excellent (E-filling scan1: r = 0.92, P < 0.001; scan2: ρ = 0.96, P < 0.001 and A-filling scan1: ρ = 0.87, P < 0.001; scan2: r = 0.99, P < 0.001). For the majority of subjects (10 out of 12), KE and EL measures showed good to strong reproducibility. However, with a wide range of agreement [intraclass correlation (ICC): 0.64–0.95] and coefficients of variation (CV) ≤ 25%. ELindex showed strong reproducibility for all 12 subjects with a strong ICC (0.94, P < 0.001) and a CV of 9%. Scan–rescan reproducibility of volumetric vorticity showed good–excellent ICCs (0.83–0.95) with CVs ≤ 11%. In conclusion, the current study shows strong–excellent in-scan consistency and overall good agreement between scans for 4D flow MRI assessment of left ventricular kinetic energy, energy loss and vorticity over diastole. However, substantial differences between the scans were also found in some parameters in two out of twelve subjects. Strong reproducibility was found in the dimensionless ELindex, which measures the amount of viscous energy loss relative to the average kinetic energy over diastole.