Purpose: To assess 24-hour fluctuations in Schlemm's canal (SC) parameters (cross-sectional area, perimeter) and intraocular pressure (IOP) and the relationship between these fluctuations in healthy individuals.Methods: SC and IOP were examined in 29 participants at 2:30, 5:30, 11:30, 17:30, and 23:30 within one day. The superior, inferior, nasal, and temporal SC quadrants were evaluated using 80-MHz ultrasound biomicroscopy.Results: SC parameters and IOP fluctuated significantly within 24 hours (all P < 0.05). After age, gender, axial length, and central corneal thickness were adjusted, compared with the baseline (23:30) value, the change in SC cross-sectional area was negatively associated with the change in IOP at 2:30, 5:30, 11:30, and 17:30 ( = -0.072[-0.094, -0.049], -0.070[-0.102, -0.038], -0.046[-0.079, -0.013], and -0.033[-0.062, -0.004], respectively; P < 0.001, < 0.001, = 0.009, and = 0.028, respectively). The nasal (175.6 36.0 pixels) and inferior (174.8 +/- 36.0 pixels) SC cross-sectional areas were significantly larger than the superior area (156.2 +/- 27.1 pixels) (P = 0.018 and 0.048, respectively) at 23:30. The observable SC proportion did not change among the quadrants or measurement time points (all P > 0.05).Conclusions: SC cross-sectional area fluctuated throughout the day and was negatively associated with changes in IOP.