Effects of a Musical Intervention on Hormonal and Inflammatory Biomarkers in Mechanically Ventilated Critically Ill Patients
Phase: NA
Conditions: Sedation, Critical Illness, Respiration, Artificial, Inflammation, Stress, Physiological
Sponsor: University of Oviedo

Study Protocol 
 
Official Title: Effects of a Musical Intervention on Hormonal and Inflammatory 
Biomarkers in Mechanically Ventilated Critically Ill Patients 
 
NCT Number: NCT06120660 
 
Sponsor: University of Oviedo 
 
Principal Investigator: Alba Maestro‑González, PhD 
 
Site: Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain 
 
Protocol Version/Date: v1.0 — 10 march 2023 
 
Statistical Analysis Plan (SAP): Included in Section 3.8  
 
 

3.1. Study Type 
Randomized, controlled, parallel-group clinical trial with blinded participants, care 
providers, and outcomes assessors. Patients were under continuous sedation, 
which ensured participant blinding. The investigator placing the headphones was 
not blinded due to the nature of the intervention. 
 
3.2. Study Setting 
The study was conducted in the General (Polivalent) Intensive Care Unit (ICU) of the 
Hospital Universitario Central de Asturias (HUCA), a major tertiary public hospital 
in Oviedo, Spain. HUCA is a modern, highly specialized center with advanced 
technological infrastructure, extensive ICU capacity, and multidisciplinary teams 
experienced in managing severe, complex critical illness. The polivalent ICU 
includes 44 beds distributed across general critical care and neuro-trauma units 
and provides comprehensive monitoring and life support for patients with 
multiorgan failure, high-risk postoperative complications, severe trauma, and 
complex infectious and medical conditions. 
 
3.3. Population and Sample 
3.3.1. Study Subjects 
Inclusion Criteria: 
• 
Adults aged 18 years or older. 
• 
Admission to HUCA’s polivalent ICU for at least 24 hours. 
• 
Intubated and receiving invasive mechanical ventilation. 
• 
Presence of an arterial catheter and/or central venous catheter. 
• 
Sedation level between BIS 40–60 or an equivalent level on the RASS scale 
(−3 to −4). 
• 
Absence of pain at baseline and during the intervention, defined as a CPOT 
score of 0. 
• 
No prior exposure to music therapy or similar interventions during the current 
hospital stay. 
• 
Informed consent obtained from the patient’s legally authorized 
representative within 72 hours of presentation. 

Exclusion Criteria: 
• 
Severe neurological disease or injury incompatible with study participation. 
• 
Severe psychiatric illness such as psychosis or schizophrenia. 
• 
Suspected or confirmed drug or alcohol intoxication, overdose, withdrawal, 
or abstinence. 
• 
Hearing impairment that limits perception of the auditory intervention. 
• 
Cranial injuries preventing safe placement of headphones. 
• 
Limitation of therapeutic effort, expected death within 24 hours, or 
confirmed brain death. 
• 
Current treatment with systemic corticosteroids. 
• 
Requirement for vasoactive drugs above 0.1 mcg/kg/min. 
• 
Refusal by the responsible attending physician. 
 
Sample Size: 
Using a pilot sample of 40 patients, a between-group difference of 1.32 units in 
serum cortisol and standard deviations of 2.86 (control) and 1.59 (intervention) were 
assumed. With 80% power and alpha = 0.05, 50 subjects per group were required. 
After adjusting for a potential 20% loss rate, the final sample size required was 63 
patients per group (126 total). 
 
3.4. Study Timeline 
Data collection occurred from 1 October 2023 to 28 June 2025. 
 
3.5. Instruments 
A study-specific case report form (CRF) was created to collect all relevant variables. 
 
3.5.1. Musical Piece 
A licensed music therapist composed a 30-minute audio piece intended to promote 
mental and physical relaxation in sedated ICU patients. The piece was reproduced 
twice consecutively, for a total session duration of 60 minutes. It was characterized 
by a soft musical dynamic (“piano”), slow tempo, two coordinated melodic lines 

(piano, synthesizer, ocean drum, acoustic guitar, soft vocals), and background 
nature sounds such as ocean waves, birds, fire crackling, and rain. The composition 
was based on a stable harmonic structure in C major, with predictable modulations 
to A minor, F, G, and occasional D minor. A diatonic scale was used to reinforce 
calmness and reduce cognitive load. 
 
3.5.2. Headphones 
The auditory intervention used Sony WH-1000XM4 noise-cancelling headphones 
with 40-mm diaphragms, extended frequency response (~40 kHz), integrated 
digital-to-analog converter with amplifier, and real-time processing to cancel 
ambient noise using external microphones and phase-inversion. This provided 
stable and high-quality sound despite the ICU environment. 
 
3.6. Variables 
Physiological variables were recorded every 10 minutes during the intervention. 
 
Demographic and Clinical Variables: 
• 
Age, sex, primary diagnosis at ICU admission. 
• 
APACHE II severity score. 
• 
Days on invasive mechanical ventilation at the time of intervention. 
• 
Ventilatory mode and parameters, including FiO2. 
• 
Sedatives, analgesics, and neuromuscular blockers, with dose and infusion 
rate. 
Physiological Parameters: 
• 
Systolic and diastolic arterial blood pressure measured via arterial catheter. 
• 
Heart rate via continuous ECG monitoring. 
• 
Respiratory rate from the ventilator. 
• 
Oxygen saturation (SpO2). 
• 
Level of sedation measured by BIS or RASS. 
Biomarkers: 
• 
Serum cortisol (primary biomarker), measured immediately before and after 
the intervention via arterial line. 

• 
Serum prolactin and interleukin-6 (IL-6), measured at the same time points. 
 
3.7. Procedures 
Approvals were obtained from the Ethics Committee of the Principality of Asturias 
and HUCA administration. ICU clinical staff were informed about study procedures. 
Patient screening involved review of clinical records and bedside nurse 
consultation. Legally authorized representatives received written and verbal 
explanation, and informed consent was obtained prior to enrollment. 
A coded identification number was assigned to each participant for confidentiality. 
 
3.7.1. Randomization and Allocation 
Participants were randomized to the music intervention group or the control group 
(quiet rest without headphones) using a computer-generated randomization list. 
The principal investigator remained unaware of allocation until consent was 
obtained. 
Blinding included participants (sedated), bedside care providers, and laboratory 
personnel analyzing biomarker samples. 
 
3.7.2. Intervention 
The process consisted of three stages: 
1. Pre-intervention (baseline). 
2. Intervention: 60-minute session of music intervention (intervention group) or 
quiet rest (control group). 
3. Post-intervention (return to usual care). 
Pain was verified using CPOT = 0 before beginning. BIS monitoring was applied when 
necessary; patients outside target sedation levels (BIS 40–60 or RASS −3 to −4) were 
excluded. Sessions occurred between 16:00 and 17:00 to align with nursing 
workflows and circadian cortisol patterns. 
Baseline arterial blood samples for cortisol, prolactin, and IL-6 were taken at 15:55. 
Physiological parameters and sedative infusion data were recorded. Headphones 
were placed. The investigator remained silently at bedside during the session. Any 
agitation or discomfort led to termination of the session and documentation. 
Post-intervention arterial blood sampling was then performed, and physiological 
variables were recorded every 10 minutes during the entire session. 

 
3.8. Statistical Analysis 
Analyses followed an intention-to-treat approach, with per-protocol sensitivity 
analyses. Continuous variables were summarized using mean ± SD or median [IQR]; 
categorical variables using frequency and percentage. 
Baseline differences were analyzed using Student’s t-test or Mann–Whitney U test 
for continuous variables and chi-square or Fisher’s exact test for categorical 
variables. 
Primary analysis focused on the change in serum cortisol using linear mixed-effects 
models with fixed effects for group, time, and group × time interaction, and random 
intercepts for subjects. Prolactin and IL-6 were analyzed similarly as key secondary 
outcomes. Non-parametric tests, including the Friedman test and Dunn post-hoc 
tests, were applied where model assumptions were violated. 
Physiological trajectories were analyzed using repeated-measures mixed models 
incorporating the 10-minute time intervals. 
Correlations were assessed using Pearson or Spearman coefficients. Multiple 
comparisons for secondary outcomes were controlled using the false discovery rate 
when necessary. 
The significance threshold was 0.05. Analyses were performed using R version 
4.4.3. 
 
3.9. Ethical Considerations and Funding 
The study adhered to the Declaration of Helsinki, the Belmont Report, CIOMS 
Guidelines, and Spanish Organic Law 3/2018 on data protection. Ethics approval 
was obtained from the Ethics Committee of the Principality of Asturias (code 
2022.537). The trial was registered at ClinicalTrials.gov (NCT06120660). Written 
informed consent was obtained from each legally authorized representative prior to 
participation. Confidentiality was ensured through coded identifiers and restricted 
data access. 
Funding: 
This study received no institutional funding from the University of Oviedo. 
Financial support was provided exclusively by the Spanish Society of Intensive and 
Coronary Care Nursing (SEEIUC) through the “SEEIUC Investiga” Grant 2023 (5th 
Edition), with a total amount of €4,000, awarded to the project “Efectos de la 
intervención musical en el estrés del paciente crítico.”