Blood sampling procedures are the most common uncomfortable and painful events experienced by patients. It can be a potential and actual unwanted consequence during diagnostic and therapeutic procedures. Pain is a major problem with negative consequences for patients and health care systems [1]. Acute pain caused by the blood sampling process is recognized as an uncontrolled and common procedure in the medical field. Its occurrence is associated with discomfort, avoidance behaviors, physiological body changes, increased sensitivity to pain, fear and avoidance of medical care [2], [3]. Its poor management leads to increased wastage of treatment work, time and resources, and ultimately leads to a decrease in patient satisfaction [4].

Currently, several pharmacological and non-pharmacological methods have been proposed to reduce acute pain, including the use of local anesthetics, distraction techniques, coughing, Valsalva maneuver, electrical stimulation, ice therapy, and balloon inflation [5]. Pharmacological approaches to pain management are usually aggressive, involving the use of multiple drugs and local anesthetics [6]. Local anesthetics are commonly used to reduce the pain of blood sampling procedures. However, many studies reported the pain caused by anesthetic injection, occurrence of delayed side effects, and skin complications such as erythema, allergic reactions, vasoconstriction, and long time to onset of effect as factors that doubt their use [7], [8], [9]. Considering that pain management is a professional principle and a moral obligation for health care providers, it is necessary to use appropriate and effective interventions to reduce pain caused by various processes.

Many local diagnostic and therapeutic invasive procedures can stimulate peripheral and cutaneous nociceptors. Cutaneous sensory neurons that express nociceptors have vanilloid1 (TRPV1) channels and sodium voltage-gated channels in free nerve endings for pain transmission [10]. These channels are activated by various painful stimuli, which causes the entry of calcium and sodium ions and finally depolarization and generation of action potential [11], [12]. Pain signals are sent to the spinal cord by Aδ and C nerve fibers and then go to different parts of the thalamus through the spinothalamic pathways and are finally processed and understood in the cerebral cortex [13]. Recent clinical literature has shown that deep brain stimulation of the thalamic structure in the delta, theta, and gamma frequency bands up to 130 Hz modulates neural oscillations of pain caused by brain lesions [14]. In another study, it was reported that the stimulation of nociceptive neurons in posterior-ventral nuclei of the thalamus with high frequencies of about 200 Hz can effectively reduce some consequences related to peripheral neuropathic pain [15]. Sound is one of the most common environmental stimuli to which living organisms are constantly exposed at different frequencies. Applying low-frequency input stimuli to the brain via the thalamo-cortical pathway can contribute to complex functional networks throughout the brain [16]. The thalamus is considered as a pathway of pain perception for further studies based on its special interaction with the cerebral cortex and its strong response to acute or chronic pain [17]. Therefore, targeting the pathways of pain transmission and the activity of the thalamus, as a relaying center, can lead to the modulation of pain perception. Stimulation of other sensory inputs to the central nervous system can be one of the pain modulating mechanisms. Pain perception can be altered by other sensory inputs, including sound. The interaction between sound and pain reduces pain perception [18]. Evidence has shown that auditory pathways in terms of intensity and frequency can reduce pain sensitivity by interacting with pain pathways [19]. Previous literature has shown that low-frequency sound stimulation (40–120 Hz), delivered via a transducer directly to the body, is effective in reducing pain [20]. Physiological mechanisms of sound-induced analgesia may be partially mediated by endogenous opioid release pathways [21], [22]. Previous researches have assessed the effects of combining different frequencies, in the form of music or sound, with high and low sound levels on the pain level [23], [24]. Music is caused by the vibration of multiple frequencies [25]. But recent studies have determined that the pain-relieving effect comes from the sound rather than the musical form [26]. Therefore, identifying the nature and characteristics of sound frequencies can provide new clinical evidence.

On the other hand, today's interventions to reduce acute pain caused by diagnostic and therapeutic procedures such as needle insertion are performed locally and through the skin, but the insertion of the needle tip into the deeper areas of the tissues is accompanied by an increase in deep traumatization of the tissue (subcutaneous layers and muscles). This event causes more stimulation of pain receptors, while most of today's anesthesia procedures are limited to local areas of the skin Therefore, it seems necessary to identify more effective analgesic processes with weakening effects in the pain transmission pathway or pain processing area in the central nervous system. The current study was designed with the aim of investigating the physioacoustic effects of low acoustic frequencies on the pain level in the processes of venous and arterial blood sampling.