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Background: Immunization is a medical procedure many children frequently undergo. As it involves pain, it is important to develop effective pain relief methods that are applicable from infancy. Objective: To clarify the pain-relieving effect of music from speakers on preschoolers during immunization. Methods: A randomized controlled trial using sex-based stratified randomization was conducted at a Japanese pediatric clinic between October and December 2016, involving 48 preschoolers aged 5 -6. Among these, 25 listened to music ("Twinkle Twinkle Little Star" played by a music box) from speakers before, during, and after influenza immunization (Music group), while the other 23 were not treated with musical intervention (Control group), with the following outcomes: primary: the Face Scale score; and secondary: the heart rate (HR) and oxygen saturation (SpO 2 ) and salivary alpha amylase (sAA) levels. Results: The Face Scale score and HR of the Control group immediately after influenza immunization were significantly higher than their baseline values (P < 0.0001, P < 0.05). There were no significant differences between the Music and Control groups in the Face Scale score, HR, or SpO 2 level immediately after influenza immunization or in the sAA level 10 minutes after it. Conclusion: Influenza immunization was painful for preschoolers. When based on the Face Scale score, HR, SpO 2 and sAA levels as indices to measure immunization-related pain, music did not show pain-relieving effects.

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