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Introduction: With the increasing demand for aesthetics, the popularity of Vacuum-formed Retainers (VFRs) has been on the rise. Bisphenol-A (BPA) is added to the polymers to make them clear and tough. BPA is identified as an endocrine disruptor linked to developmental defects, infertility, cardiovascular disease and cancer. The release of BPA from various orthodontic materials, including VFRs, has been reported infrequently and with variable results. Aim: To assess the effect of immersing VFRs in distilled water on BPA release in the saliva of patients wearing VFRs. Materials and Methods: This was a single-centre prospective randomised clinical trial. A total of 28 patients aged between 15 and 25 years, who had completed their fixed orthodontic treatment in 2021 at Bharati Vidyapeeth Deemed to be University (BVDU), Dental College and Hospital, Sangli, Maharashtra, India, were selected for this in-vivo study. Patients were randomly divided into two groups. Group I patients received VFRs without immersion in water before delivery, while Group II patients received VFRs that were immersed in distilled water at 37˚C for 24 hours before delivery. Four samples of unstimulated whole saliva were collected across four different time intervals: T0 (before VFR delivery), T1 (after one hour of wearing retainers), T2 (after one week of wearing retainers) and T3 (after three weeks of wearing retainers). These samples were tested for BPA release using HPLC analysis. For numerical continuous data following a normal distribution, intergroup comparison was conducted using a one-way Analysis of Variance (ANOVA) test and intra-group comparisons were performed using a paired t-test. An alpha error of 5% and a power of 80% were maintained, with p