Recent research indicates that brief periods (60 min) of monocular defocus lead to small but significant changes in human axial length. However, the effects of longer periods of defocus on the axial length of human eyes are unknown. We examined the influence of a 12 h period of monocular myopic defocus on the natural daily variations occurring in axial length and choroidal thickness of young adult emmetropes. A series of axial length and choroidal thickness measurements (collected at ∼3 hourly intervals, with the first measurement at ∼9 am and the final measurement at ∼9 pm) were obtained for 13 emmetropic young adults over three consecutive days. The natural daily rhythms (Day 1, baseline day, no defocus), the daily rhythms with monocular myopic defocus (Day 2, defocus day, +1.50 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, recovery day, no defocus) were all examined. Significant variations over the course of the day were observed in both axial length and choroidal thickness on each of the three measurement days (p < 0.0001). The magnitude and timing of the daily variations in axial length and choroidal thickness were significantly altered with the monocular myopic defocus on day 2 (p < 0.0001). Following the introduction of monocular myopic defocus, the daily peak in axial length occurred approximately 6 h later, and the peak in choroidal thickness approximately 8.5 h earlier in the day compared to days 1 and 3 (with no defocus). The mean amplitude (peak to trough) of change in axial length (0.030 ± 0.012 on day 1, 0.020 ± 0.010 on day 2 and 0.033 ± 0.012 mm on day 3) and choroidal thickness (0.030 ± 0.007 on day 1, 0.022 ± 0.006 on day 2 and 0.027 ± 0.009 mm on day 3) were also significantly different between the three days (both p < 0.05). The introduction of monocular myopic defocus disrupts the daily variations in axial length and choroidal thickness of human eyes (in terms of both amplitude and timing) that return to normal the following day after removal of the defocus.