INTRODUCTION: A major goal in neonatal medicine is to reduce stress as much as possible in routine care. Bathing is one of the important routine cares for neonates, but it makes a big environmental change for them. We aimed to examine whether water temperature, room temperature, and position changes in tub bathing serve as noxious stimuli to neonates. METHODS: This prospective trial was performed in full-term and non-low-birth-weight neonates admitted to the hospital between July 2020 and March 2021. Those with underlying diseases, fetal distress, infection, and other medical conditions were excluded. Measurements were taken during the neonates' first tub bath since birth, which was performed by a trained nurse. Changes in regional oxygen saturation (rSO2), determined using near-infrared spectroscopy, and water and room temperature, were examined at five different time points: upon entering the bath, head washing, position change, exiting the bath, and during the 3 min after bathing. RESULTS: In total, 17 neonates were analyzed. No changes in rSO2 due to head washing or position change were observed; however, rSO2 significantly decreased upon entering (78.5 ± 4.1% vs. 75.7 ± 4.1%, p < 0.001) and exiting the bath (75.8 ± 5.7% vs. 74.4 ± 5.4%, p < 0.04). The rate change in rSO2 upon entering the bath showed a significant inverse correlation with water temperature (r =  - 0.53, p < 0.03), and there were no significant correlations between rSO2 and water or room temperature upon exiting the bath. There was no change in body skin temperature before and after bathing, but rSO2 gradually decreased during the 3 min after bathing. CONCLUSIONS: Neonates may perceive certain temperatures during bathing as noxious stimuli. Therefore, methods to minimize stress associated with bathing should be implemented to reduce the difference between water temperature and room temperature during bathing. TRIAL REGISTRATION: This trial has been registered at UMIN repository with the trial number UMIN000041045 ( https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000046500 ). The date of the final dataset was April 01, 2021.