Melatonin and Cortisol concentration Before and After CPAP Treatment o

Background

Obstructive Sleep Apnea (OSA) is a widespread sleep disorder characterized by recurrent episodes of upper airway obstruction during sleep, resulting in intermittent hypoxia, sleep fragmentation, and excessive daytime sleepiness.^1,2 Globally, OSA affects approximately 8.5–18% of the population, with a higher prevalence among specific groups, such as commercial truck drivers (29–35.5%), highlighting its public health significance.^3,4 In addition to short-term effects on quality of sleep, OSA is linked with systemic complications such as cardiovascular diseases, metabolic disorders, and neurocognitive impairment.⁴ One of the major mechanisms behind this impact involves dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis and circadian rhythm. Research has shown that sleep deprivation and stress can disrupt normal cortisol secretion patterns, leading to alterations in the hypothalamic-pituitary-adrenal (HPA) axis activity and increased vulnerability to mood disorders.⁵ Furthermore, excessive exposure to light at night, such as that emitted from digital screens, has been linked to circadian misalignment and suppression of melatonin, thereby exacerbating endocrine and metabolic disturbances.⁶

Cortisol, a critical glucocorticoid, modulates metabolism, stress responses, and the sleep-wake cycle, typically peaking in the morning (0.36–7.56 ng/mL) and reaching a nadir at midnight (0.54–2.28 ng/mL).⁷ In contrast, melatonin, secreted by the pineal gland, governs circadian timing and is primarily released in the evening to facilitate sleep onset (<3 pg/mL during the day and up to 100 pg/mL a night).^8,9 In OSA, sleep fragmentation and hypoxia may disrupt these hormonal rhythms. Preliminary evidence suggests that patients with OSA exhibit delayed cortisol peaks, while melatonin secretion shows variable responses.¹⁰ Although Continuous Positive Airway Pressure (CPAP) is the gold-standard treatment for moderate to severe OSA and improves outcomes such as daytime alertness and cognitive function,¹¹ its impact on melatonin and cortisol concentration – and thus on circadian rhythm restoration- remains underexplored.¹²

This knowledge gap is particularly pressing given the roles of these hormones in sleep regulation and overall health.^13–15 In Vietnam, where OSA affects 8.5% of the population, the condition remains underdiagnosed and lacks biomarker-guided management.¹⁶ Understanding the endocrine effects of CPAP therapy could optimize treatment strategies and help mitigate accident risks.¹⁷ This study investigates the salivary melatonin and cortisol concentrations in OSA patients before and after CPAP treatment, aiming to elucidate the endocrine effects of OSA therapy and underscore the importance of early diagnosis and intervention to improve patient outcomes.

Subjects and Methods

Study Design and Participants

This prospective before-and-after study was conducted at the University Medical Center Ho Chi Minh City, and CHAC clinic between November 2022 and August 2024. The study aimed to evaluate the changes in sleep-related neuroendocrine biomarkers and psychophysiological performance in patients diagnosed with obstructive sleep apnea (OSA) and treated with continuous positive airway pressure (CPAP).

General characteristics of the study subjects included age (years), gender, education level, marital status, and body mass index (BMI). Additional clinical and behavioral variables included the Apnea–Hypopnea Index (AHI), minimum peripheral oxygen saturation (SpO₂), and lifestyle factors such as smoking status, alcohol consumption, caffeine and tea intake, and sleep duration. Psychological status was assessed using the Depression, Anxiety, and Stress Scales (DASS-21). Hormonal biomarkers, including salivary melatonin and cortisol concentrations, were measured at two time points (morning and afternoon) to assess circadian rhythm alterations in relation to obstructive sleep apnea (OSA) and the effects of CPAP therapy.

The study adhered to the Declaration of Helsinki and was approved by the institutional review board of the University of Medicine and Pharmacy at Ho Chi Minh City (Approval Number: 657/HĐĐĐ-ĐHYD, dated 18/08/2022). Written informed consent was obtained from all participants.

Participants

Eligible participants were adults (≥18 years old) with a confirmed diagnosis of OSA, defined by an apnea-hypopnea index (AHI) ≥5 events/hour and clinical symptoms of OSA, or AHI ≥15 events/hour regardless of symptoms. All patients selected CPAP as their treatment method and provided informed consent. Patients were excluded if they had other sleep disorders (eg, insomnia, central sleep apnea), uncontrolled comorbidities (eg, COPD, heart failure, neuromuscular disease), recent shift work or jet lag within one week prior to each visit, previous OSA-specific treatment, or were pregnant during the study. The entire recruitment process is illustrated in Figure 1.

Figure 1 Study flowchart. Abbreviations: OSA, Obstructive Sleep Apnea; CPAP, Continuous positive airway pressure; ELISA, Enzyme-Linked ImmunoSorbent Assay.

Data Elements and Laboratory Methods

A total of 59 patients were enrolled at baseline (before treatment), with 27 completing the follow-up assessment after 8 weeks of CPAP treatment. Participants were selected consecutively from patients diagnosed with OSA who opted for CPAP and met the eligibility criteria. The post-treatment sample size was reduced to 27 participants as many patients were unable to return for follow-up visits within the study timeframe, primarily due to scheduling and logistical limitations.

At Visit 0 (screening), patients diagnosed with obstructive sleep apnea (OSA) were provided counseling on available treatment options, including lifestyle modifications, continuous positive airway pressure (CPAP), mandibular advancement devices, and upper airway surgery. Patients who chose CPAP therapy were further assessed for study eligibility, informed about the study protocol, and provided written informed consent if they met the inclusion criteria. Visit 1 (baseline) involved the collection of baseline data and salivary biomarker sampling. Visit 2 (follow-up), conducted eight weeks after CPAP initiation, included repetition of the assessments from Visit 1 and collection of CPAP adherence data from the device. Prior to assessments, patients were instructed to abstain from caffeine and alcohol. Medications known to affect alertness or corticosteroids were either excluded based on their half-life if used acutely, or maintained if prescribed for chronic conditions. Avoidance of screen exposure prior to sample collection was not controlled in this study.

Collection of Saliva Samples

Participants expectorated 2mL of saliva into a sterile, 15 mL polyethylene tube. Salivary samples were collected twice in the morning (8:00–9:00 AM) and afternoon (3:00–4:00 PM) after 30 minutes of fasting and 10 minutes after rinsing mouth. These collection tubes were maintained on ice, centrifuged at 10,000 rpm for 5 minutes at 4°C. The supernatants were harvested and stored at −70°C for further experiments.

Measurement of Salivary Melatonin and Cortisol

Melatonin and cortisol concentrations in saliva were quantified using Human MT (Melatonin) ELISA Kit (sensitivity: 4.688pg/mL; intra-assay CV% < 8, cat no.: MBS766108, MyBioSource, San Diego, USA) and Human COR (Cortisol) ELISA Kit (sensitivity: 0.234ng/mL, intra-assay CV% < 8, cat no.: MBS766080, MyBioSource, USA), respectively, based on competitive ELISA detection method. The microtiter plate provided in the kits was pre-coated with target capture antibody. Each standard and sample was added into the microtiter plate (50 µL/well), followed immediately by 50 µL of Biotin-labeled Antibody Working Solution, and the plate was incubated at 37°C for 45 min. After 3 washes, 100 µL of HRP-Streptavidin Conjugate Working Solution is added, followed by a 30-min incubation at 37°C. The plate was then washed 5 times before adding 90 µL of 3,3′,5,5′-tetramethylbenzidine substrate solution and incubating for 10–20 min at 37°C in the dark. The reaction was stopped by adding 50 µL of Stop Solution to each well. Finally, the absorbance was measured at 450 nm, and sample concentrations were calculated from a standard curve, adjusting for dilution factors when applicable.

Calculation and Definition

The primary outcomes of this study included the concentrations of salivary melatonin and cortisol before and after CPAP treatment.

Independent variables included demographic characteristics (age, sex, education level, marital status); body mass index (BMI); and lifestyle factors (smoking status, alcohol consumption, tea and coffee intake, and timing of last intake of alcohol or caffeine). Sleep-related metrics comprised nighttime and daytime sleep durations.

Mental health status was assessed using the Depression, Anxiety, and Stress Scales (DASS-21). Clinical variables included the Apnea-Hypopnea Index (AHI), which was categorized into mild (5 ≤ AHI < 15), moderate (15 ≤ AHI < 30), and severe (AHI ≥ 30); minimum and average peripheral oxygen saturation (SpO₂).

CPAP adherence is defined as using the CPAP machine for at least 4 hours per night on at least 70% of nights, over a 30-day period before visit 2, all data extracted from CPAP device software.

Statistical Analysis

Categorical variables were presented as frequencies and percentages, while continuous variables were presented as means and standard deviations (SD) and medians (interquartile ranges – IQR). Differences between proportions were tested using chi-square and alternative Fisher’s tests under the condition of <5%. Quantitative variables with normal distribution were compared between two independent groups (before and after treatment) using an independent samples t-test, while the non-parametric Wilcoxon rank-sum test was applied for variables that did not follow a normal distribution.

To assess the changes in hormone concentrations (melatonin and cortisol) before and after treatment in the same group of subjects, the Wilcoxon signed-rank test was used. Spearman rank correlations were used to assess the monotonic associations between hormone concentrations (pre- and post-treatment) and clinical and behavioral variables such as daytime sleepiness, sleep duration, caffeine consumption, psychological symptoms, and respiratory index. The threshold for statistical significance was set at p < 0.05. All analyses were performed using R software version 4.4.2.

Results

Baseline Characteristics

The demographic characteristics of patients at baseline (n = 59) and of those who completed the 8-week CPAP treatment (n = 27). The majority of participants in both groups were male (78.0% at baseline; 85.2% post-treatment). The mean age of patients was 48.4 ± 13.3 years before treatment and 45.1 ± 13.2 years among those who completed follow-up. Mean BMI values were 28.3 ± 5.2 and 29.5 ± 5.3, respectively. Regarding educational background, most participants had attained university or higher education levels. Specifically, 42.4% of baseline participants and 59.3% of those post-treatment had completed university education. A small proportion had postgraduate education (6.8% and 7.4%, respectively). Marital status, most participants were married in both groups (74.6% and 74.1%, respectively), while single individuals accounted for 16.9% and 18.5% of the baseline and post-treatment groups. Office workers represented the largest group in both cohorts (33.9% at baseline and 51.9% post-treatment). No significant baseline demographic or clinical differences were observed between patients completing CPAP treatment (n = 27) and the original, indicating the post-treatment group remained representative of the initial population (Table 1), reducing potential selection bias concerns (Table 1).

Table 1 Demographic Characteristics of Patients According to the AHI

Power analysis (paired melatonin, n=27). Using the observed variability (SD pre =52.5, SD post =37.7) and correlation r=−0.3538, the SD of within-subject differences is SDΔ=74.7. For a two-sided α=0.05 paired design, the study has ~80% power to detect a mean pre-to-post change of 40.3 units (dz≈0.54); ~88% for 44.8 (dz=0.60); and >95% for 52.3 (dz=0.70). Thus, with 27 completers, the study is powered to detect moderate within-subject changes; adequacy depends on the prespecified clinically meaningful change.

The proportions of medication use, smoking, alcohol consumption, daytime sleepiness, daily coffee or tea intake, sleep duration, and psychological disorders did not differ significantly between the two groups (Table 2).

Table 2 Medication, Lifestyle, Time Sleep, Mental Health, and Paraclinical Factors of the Before and After Treatment Subjects

Before treatment for OSA, melatonin and cortisol exhibited substantial interindividual variability. The mean melatonin was 80.80 ± 52.48 pg/mL, with higher values observed in the afternoon (108.76 ± 81.30 pg/mL) compared to the morning (52.85 ± 41.31 pg/mL). The mean cortisol concentration was 7.58 ± 5.45 ng/mL, with higher concentrations in the morning (10.12 ± 6.98 ng/mL) than in the afternoon (5.04 ± 4.69 ng/mL), reflecting a normal circadian pattern (Table 3).

Table 3 Melatonin and Cortisol Concentration of the Study Groups

After treatment, melatonin decreased to 66.88 ± 37.7 pg/mL, and the morning–afternoon difference narrowed (53.11 vs 80.64 pg/mL), suggesting a trend toward stabilization. Cortisol concentration increased slightly to 9.08 ± 7.96 ng/mL, while maintaining a physiological distribution with higher concentration in the morning than in the afternoon (Table 3).

While overall melatonin levels showed a trend toward reduction from 74.88 to 59.80 pg/mL (p=0.313), and afternoon melatonin concentrations decreased from 120.34 to 71.07 pg/mL (p=0.130), these changes did not reach statistical significance. Cortisol levels remained relatively stable across all time points, with no significant differences observed between pre- and post-treatment measurements (Table 4).

Table 4 Comparison of Melatonin and Cortisol Concentration in the Treatment Group (n=27)

Treatment may reduce afternoon melatonin concentration, potentially helping to restore the disrupted circadian rhythm observed in patients with OSA. Additionally, cortisol concentrations appear to become more regulated, particularly with a slightly increase in afternoon concentration, aligning with the normal physiological pattern of higher cortisol in the morning and lower concentration in the afternoon (Figure 2).

Figure 2 Patient-specific changes in melatonin and cortisol before and after treatment.

Melatonin and cortisol concentrations before and after treatment were further detailed by gender, age, smoking status, alcohol use, and obesity. These subgroup analyses were exploratory due to small sample sizes (n ≈ 6–8 per group). Melatonin tended to decrease, while cortisol tended to increase after treatment, especially in the non-alcoholic group, suggesting that the two hormones responded in opposite directions. Melatonin changed most strongly in the non-obese group, decreasing from 87.83 (26.93–148.73 pg/mL) before treatment to 35.43 (26.83–44.03 pg/mL) after treatment. In males, melatonin also decreased significantly from 91.90 to 57.01 pg/mL. Some groups increased slightly after treatment, such as females (from 67.96 to 80.40 pg/mL), smoking, and those ≤55 years. Cortisol changed most dramatically in the non-alcohol group, increasing from 3.70 (0.97–11.77 ng/mL) to 11.81 (4.2–16.42 ng/mL) after treatment. In addition, the non-smoking and >55 years also had a slight increase in median cortisol, but not as much as the nondrinker group (Table 5).

Table 5 Melatonin and Cortisol Concentration Before and After Treatment by Gender, Age, Smoking Status, Alcohol Use, and Obesity (n=27)

Figure 3 illustrates the interaction effects between treatment time (before vs after CPAP) and subgroup characteristics (gender, age, smoking status, alcohol use, and obesity) on salivary melatonin and cortisol levels. The linear mixed-effects model did not identify any statistically significant interaction effects (all p-values > 0.05), suggesting that the changes in hormone concentrations after CPAP treatment were consistent across subgroups. While some numerical differences were observed, these did not reach statistical significance, indicating that demographic and behavioral factors did not significantly modify the hormonal response to CPAP therapy.

Figure 3 Comparisons of melatonin and cortisol concentration before and after treatment within each gender (A), age group (B), smoking status (C), alcohol use (D), and obesity (E) (by Linear Mixed-Effects Model.

The number of cups of tea consumed per day was significantly negatively correlated with melatonin concentration before treatment (rho = –0.43, p<0.05). The remaining variables, including coffee consumption, sleep durations, psychological symptoms (depression, anxiety, stress), AHI, SpO₂ indices, and compliance, showed no statistically significant correlations with melatonin or cortisol concentration (Table 6).

Table 6 Computed to Assess Melatonin and Cortisol Concentration Before and After Treatment with Various Clinical and Behavioral Variables

Objective CPAP adherence showed no significant correlation with changes in salivary melatonin or cortisol levels (all p > 0.05). Variability in adherence across participants did not appear to explain the observed hormonal patterns.

Discussion

This study investigated changes in salivary melatonin and cortisol concentrations before and after Continuous Positive Airway Pressure (CPAP) therapy in patients with OSA. Although none of the observed differences reached statistical significance, several physiological trends were evident and are consistent with prior findings.

At baseline, in the treatment group, OSA patients exhibited abnormally elevated afternoon melatonin levels (120.34 pg/mL), which is unusual because healthy individuals typically have low daytime melatonin levels. After CPAP treatment, we observed a nonsignificant trend toward a decrease in afternoon melatonin levels (to 71.07 pg/mL, p = 0.130), which may represent normalization of the disrupted circadian rhythm pattern, although statistical significance was not reached. Post-treatment melatonin concentrations showed a decreasing trend, especially in the afternoon, suggesting a narrowing of the diurnal variation. This may reflect a partial restoration of circadian rhythm, which is frequently disrupted in OSA due to sleep fragmentation, intermittent hypoxia, and sympathetic overactivation.¹⁸ A study by Zirlik et al showed that after 3 months of CPAP treatment, the melatonin peak shifted to 2 a.m. in patients with preexisting melatonin secretion disorders.¹⁹ While CPAP may contribute to normalizing melatonin secretion patterns in some studies,²⁰ our study lacked sufficient power to confirm such an effect.

Our study suggests that higher tea consumption may be associated with lower melatonin concentration, possibly due to the effect of caffeine in tea on melatonin production. Consuming caffeine in the evening may reduce melatonin secretion at night, leading to poorer sleep.²¹ Other research suggests that caffeine may slow down melatonin circadian rhythms, affecting the body’s natural sleep-wake cycle.²²

Cortisol concentrations showed a slight overall increase post-treatment, with morning values remaining higher than those in the afternoon. The systematic review by Tomfohr LM et al analyzed 15 studies investigating the relationship between OSA and cortisol concentration, as well as the impact of continuous CPAP therapy on this hormone.²³ The results showed that only one study reported a significant difference in cortisol concentration between OSA patients and controls, and only two studies demonstrated significant changes in cortisol concentration before and after CPAP treatment. However, due to inconsistencies in sampling methods and timing of cortisol measurement, current evidence remains insufficient to draw definitive conclusions about the association between OSA and cortisol alterations, or the efficacy of CPAP in modulating cortisol concentration.²³

Several studies have demonstrated that sleep quality influences morning cortisol concentrations. Hansen AM et al reported that poorer sleep quality over the preceding month was associated with lower cortisol concentration upon awakening.²⁴ Conversely, Backhaus J et al found that patients with insomnia exhibited significantly higher morning cortisol concentrations compared to healthy controls.²⁵ These contrasting findings highlight the complex relationship between sleep disturbances and HPA axis regulation, suggesting that different types of sleep impairment may differentially affect cortisol dynamics.^24,25

In our study, eight weeks may be inadequate for complete HPA axis normalization in OSA patients. The HPA axis undergoes complex adaptations during chronic OSA, and restoration to normal patterns may require longer treatment periods. In addtion, CPAP initiation may represent a physiological and psychological stressor that temporarily elevates cortisol before eventual normalization. Our 8-week timepoint may have captured patients during this adaptation phase rather than after full acclimatization. Studies with longer follow-up periods might show the expected cortisol reduction. Therefore, hormonal biomarkers may not be suitable for early assessment of CPAP efficacy and that longer-term monitoring is necessary.

Recent evidence further supports the role of CPAP in ameliorating circadian clock disruptions. Gaspar LS et al demonstrated that OSA alters biological clock-related characteristics that differentially respond to short- and long-term CPAP treatment.²⁶ Long-term CPAP was more efficient in counteracting OSA’s impact on the clock, but the obtained results suggest that it is not fully effective.²⁶ Similarly, Gabryelska A et al indicate that circadian clock disruption among patients with OSA can be at least partially changed by CPAP application, even after one night of intervention.²⁷ Pointing to complex mechanisms involved in the circadian clock elements’ reaction to alternating oxygen levels.^26,27

Our study suggests that although the statistics were not significant, there is a biological reason for our findings regarding circadian rhythms. In patients with OSA, high afternoon melatonin levels (108.76 ± 81.30 pg/mL) are probably related to increased sleep pressure. Sleep disruption in OSA patients often increases the need for daytime sleep, causing melatonin to be released at an inappropriate time of day. After CPAP treatment, afternoon melatonin levels decreased to 80.64 pg/mL, which is consistent with the idea that better sleep reduces daytime sleep pressure. However, cortisol levels increased slightly, which was not what we expected. This may be due to the fact that 8 weeks of treatment is not enough for complete recovery, or people respond differently to CPAP, or the body’s stress system needs time to recover from long-term sleep problems.

Study Limitations

The post-treatment sample size was relatively small, reducing the statistical power to detect subtle hormonal changes. With a final sample of 27 paired observations, the study is underpowered to detect modest (<30%) hormone changes; therefore, results should be interpreted as hypothesis-generating rather than confirmatory. While salivary biomarkers offer practical circadian assessment, the chosen daytime windows may not capture nocturnal dynamics critical to OSA-related dysregulation. In our study, saliva collection is challenging at night, when melatonin secretion is at its highest (2–4 AM), and it is also challenging to manage for our patients who collect saliva at home at other times (partially reflected in the dropout rates). Another consideration is the use of salivary rather than serum samples for hormone measurement. While salivary assays offer a non-invasive and practical approach for assessing free, biologically active hormone fractions, they may be more susceptible to pre-analytical variability (eg, flow rate, contamination, sample handling) and have lower sensitivity compared to serum-based assays. These methodological differences could potentially mask subtle hormonal changes and limit the precision of circadian profiling. Moreover, although no significant hormonal changes were observed following CPAP treatment, several factors may explain these null results. The treatment duration may have been insufficient for hormonal normalization, particularly if underlying neuroendocrine disruptions require more prolonged intervention. Additionally, adherence to CPAP therapy, though monitored, was not objectively quantified in this study. Suboptimal or inconsistent use could attenuate physiological effects and mask potential improvements in circadian hormone profiles. We also acknowledge that the lack of a control group is a limitation of our study. In the data we have so far, there have been no reports of salivary cortisol and melatonin concentrations in the Vietnamese population, so there may be differences compared to studies in other ethnic groups. Future longitudinal studies with larger sample sizes with control group, objective CPAP adherence metrics, serum-based or combined biospecimen approaches, and full circadian sampling are warranted to confirm these findings and better understand the relationship between CPAP therapy and endocrine outcomes.

Although this is a small sample size study with limitations, it is the first study in Vietnam on the hormones related to circadian rhythms and OSA. With limited funding and low compliance of patients, we could only conduct the study at times that were consistent with the clinic hours of the day. With some initial trends, although not statistically significant, it can help scientists and clinicians in Vietnam in particular and the world in general have more basis to conduct larger studies in the future.

Conclusion

This preliminary study did not identify statistically significant changes in salivary melatonin or cortisol concentrations following 8 weeks of CPAP treatment in 27 patients with OSA, however, trends indicated reduced sleep pressure, including lower afternoon melatonin and higher cortisol levels. The substantial dropout rate constrains the interpretation of these findings. Further research involving larger sample sizes, extended treatment durations, and more comprehensive hormonal assessments at various times of the day is necessary to elucidate the potential role of salivary biomarkers in understanding circadian regulation in OSA.

Data Sharing Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request. However, the datasets are not publicly available due to ethical and privacy considerations involving patient confidentiality.

Acknowledgments

We would like to extend our sincere gratitude to the University Medical Center Ho Chi Minh City, and CHAC Clinic for their excellent support and cooperation in conducting this study. We would also like to thank all the patients who actively agreed to participate in this study. Thanks to Thanh Huynh Ngoc and Phuong Nguyen Thi Be for their assistance in data interpretation and manuscript editing.

Author Contributions

KBD – conceptualization, data curation, writing – original draft, writing – review and editing, project administration. NVT – conceptualization, supervision, writing – review and editing. TNB – data curation, writing – original draft. QDT – data curation, writing – original draft. HKTT – investigation (sample preservation), writing – review and editing. DDKT – investigation (sample preservation), data curation (ELISA analysis), writing – review and editing. KML – investigation (sample preservation), data curation (ELISA analysis), writing – review and editing. LPK – methodology, formal analysis, writing – review and editing. SDQ – conceptualization, methodology, supervision, writing – review and editing. All authors – literature review, writing – review and editing, approval of final manuscript, accountability for all aspects of the work. All authors agreed on the journal to which the article has been submitted. KBD and NVT are both first authors.

Funding

This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City under contract number 225/2022/HĐ-ĐHYD, dated 14/10/2022.

Disclosure

The authors declare that there are no conflicts of interest related to this study.

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