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To investigate the influence of transcytolemmal water exchange on estimates of tissue microstructural parameters derived from diffusion MRI using conventional PGSE and IMPULSED methods.Computer simulations were performed to incorporate a broad range of intracellular water life times τin (50–∞ ms), cell diameters d (5–15 μm), and intrinsic diffusion coefficient Din (0.6–2 μm2/ms) for different values of signal-to-noise ratio (SNR) (10 to 50). For experiments, murine erythroleukemia (MEL) cancer cells were cultured and treated with saponin to selectively change cell membrane permeability. All fitted microstructural parameters from simulations and experiments in vitro were compared with ground-truth values.Simulations showed that, for both PGSE and IMPULSED methods, cell diameter d can be reliably fit with sufficient SNR (≥ 50), whereas intracellular volume fraction fin is intrinsically underestimated due to transcytolemmal water exchange. Din can be reliably fit only with sufficient SNR and using the IMPULSED method with short diffusion times. These results were confirmed with those obtained in the cell culture experiments in vitro.For the sequences and models considered in this study, transcytolemmal water exchange has minor effects on the fittings of d and Din with physiologically relevant membrane permeabilities if the SNR is sufficient (> 50), but fin is intrinsically underestimated.