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In this study, the effect of lung volume on quantitative measures of lung ventilation was investigated using MRI with hyperpolarized ³He and ¹²⁹Xe. Six volunteers were imaged with hyperpolarized ³He at five different lung volumes [residual volume (RV), RV + 1 liter (1L), functional residual capacity (FRC), FRC + 1L, and total lung capacity (TLC)], and three were also imaged with hyperpolarized ¹²⁹Xe. Imaging at each of the lung volumes was repeated twice on the same day with corresponding ¹H lung anatomical images. Percent lung ventilated volume (%VV) and variation of signal intensity [heterogeneity score (H_score)] were evaluated. Increased ventilation heterogeneity, quantified by reduced %VV and increased H_score, was observed at lower lung volumes with the least ventilation heterogeneity observed at TLC. For ³He MRI data, the coefficient of variation of %VV was <1.5% and <5.5% for H_score at all lung …