Purpose: The use of vaginal immobilization balloons placed into the vagina for immobilization of tandem and ovoid (T+O) applicator during high-dose-rate (HDR) brachytherapy delivery has been used at our institution, and seems to have improved our patient comfort, decreased procedure time, and minimized applicator misplacement. We aimed to show that these balloons, while originally marketed for single-day use, are safe and maintain applicator positioning/dosimetry when left in situ overnight for treatment delivery on sequential days.Material and methods: Forty-two paired computed tomography (CT) scans from thirteen patients who underwent T+O HDR treatments on sequential days with vaginal immobilization balloons in situ overnight were retrospectively compared to calculate mean change of balloon volumes and balloon/T+O distance to bony landmarks. Dosimetric planning was retroactively performed on day 2 using CT scan of each pair, and the change in estimated radiation delivery to the bladder and rectum was compared.Results: No statistically significant overnight changes were found in balloon volumes or anterior balloon positioning. The posterior balloon shifted -0.29 ±0.46 cm (p = 0.03) to the anterior public symphysis and 0.32 ±0.50 cm (p = 0.01) to the right femoral head. The tandem shifted 0.37 ±0.39 cm (p = 0.002) to the pubic symphysis. There was no significant difference found in radiation delivered to the bladder or rectum between the paired scans.Conclusions: This study showed minimal change in balloon volumes, balloons/T+O positioning, or in radiation dose to bladder and rectum when the applicator remained overnight. These findings support that inflatable vaginal immobilization balloons remaining in situ overnight for additional HDR T+O treatments on sequential days, is safe and provides stable dosimetry.