["Acta Physiologica, Volume 242, Issue 6, June 2026. ", "\nABSTRACT\n\nAim\nEchocardiographic assessment of diastolic function in mice remains challenging because parameters translated from clinical practice are constrained by murine physiology. In particular, parameters derived from early (E) and late (A) transmitral filling velocities are frequently compromised by wave fusion at higher heart rates. Here, we quantified these limitations and evaluated an alternative method for measuring isovolumetric relaxation time (IVRT), a marker reflecting ventricular relaxation and a key element of established algorithms specific for murine diastolic function assessment.\n\n\nMethods\nIn a non‐A4C‐view‐dependent echocardiographic measurement (NAEM) approach, speckle‐tracking analysis of standard brightness‐mode (B‐mode) cine loops from the parasternal long‐axis view (PSLAX) enabled E/A ratio and IVRT assessment. Mice with unimpaired heart function, heart failure with preserved ejection fraction (HFpEF), or heart failure with reduced ejection fraction (HFrEF) (n = 8 each) underwent standard Doppler‐based and NAEM‐based measurement of E/A ratio and IVRT.\n\n\nResults\nIn mice with unimpaired heart function and HFpEF, heart rate‐dependent E and A wave fusion occurred at an estimated threshold of approximately 460 bpm, whereas advanced HFrEF led to frequent fusion independent of heart rate. NAEM‐derived IVRT showed strong agreement with the Doppler reference (r2 = 0.9339, p < 0.0001), minimal bias (−1.4 ms), and similar inter‐observer variability. In contrast, NAEM‐derived E/A ratio did not show adequate performance.\n\n\nConclusion\nThese findings further support the shift toward specific algorithms for diastolic function assessment in murine echocardiography. The validated NAEM approach supports application of such protocols without dedicated Doppler recordings by using PSLAX B‐mode cine loops, thereby lowering technical barriers for future studies and enabling robust retrospective analysis of existing datasets.\n\n"]