Abstract: Melatonin influences arterial biomechanics, and its absence could cause remodeling of the arterial wall, leading to increased stiffness. Direct effects of fentanyl on the aortic wall have also been observed previously. This study aimed to evaluate in vitro the effects of fentanyl on aortic viscoelasticity in a rat model of melatonin deficiency. It also aimed to test the hypothesis that melatonin deficiency leads to increased arterial wall stiffness. The viscoelasticity of the aortic wall was estimated in strip preparations from pinealectomised and sham-operated adult rats using the forced oscillations method. We found that melatonin deficiency in the untreated aortic wall did not significantly alter viscoelasticity. However, combined with 10-9 M fentanyl, melatonin deficiency increased the natural frequency and arterial stiffness compared to the sham-operated. Independently, fentanyl treatment decreased the natural frequency and arterial stiffness compared separately to untreated normal and melatonin-deficient preparations. The observed effects of fentanyl were neither dose-dependent nor affected by naloxone, suggesting a non-opioid mechanism. Furthermore, an independent effect of naloxone was also detected in the normal rat aortic wall, resulting in reduced arterial stiffness. Further studies are needed to clarify the underlying mechanisms, which may improve the clinical decisions for pain management and anesthesia.