Change in Salivary Cortisol Levels in Children (Aged 9–12 Years) with Obesity and Respiratory Diseases during a 28-Day Spa Treatment: A Pilot Prospective Study

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The study included 95 children at spas and 38 children from a primary school (control group). Laboratory analyses were performed at the Institute of Endocrinology in Prague. In children with obesity (n = 34), a decrease in mean cortisol levels from 3.32 to 2.36 nmol/l was observed. In children with respiratory diseases not receiving corticosteroid therapy (n = 18), cortisol levels increased from 2.68 to 3.48 nmol/l. The control group showed an increase from 3.52 to 4.02 nmol/l (n = 38). These findings highlight the relevance of salivary cortisol as a dynamic stress biomarker for pediatric rehabilitation research. The findings support the utility of salivary cortisol as a biomarker of physiological response to therapeutic and rehabilitative interventions in pediatric populations. saliva cortisol child balneology biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In recent decades, there has been growing interest in the objective assessment of spa treatment effects not only on somatic health but also on neuroendocrine and psychosocial functions. This approach is grounded in the concept of the psycho–neuro–immune–endocrine (PNIE) axis [ 1 ], which highlights the interconnectedness of the central nervous system, endocrine regulation, immune responses, and an individual’s psychological state [ 2 , 3 ]. This integrative perspective is well captured by the assertion: “The musculoskeletal system responds to all external and internal dysfunctions of the body in an integrative manner, and most of them can be influenced or eliminated through the musculoskeletal system.” [ 4 ] Spa treatment, which combines physical activity, dietary regulation, and exposure to stimulating natural environments, provides a natural framework for studying complex adaptive mechanisms. Previous studies in adult populations undergoing structured spa or rehabilitation treatments have shown that such interventions may modulate stress physiology, leading to reduced cortisol levels and improved neuroendocrine regulation. These effects are often accompanied by enhancements in subjective well-being, physical fitness, and metabolic health. Within this context, salivary cortisol—an established non-invasive biomarker of hypothalamic–pituitary–adrenal (HPA) axis activation—provides an accessible index of stress-related physiological states. In pediatric populations, the measurement of salivary cortisol is particularly advantageous due to its non-invasiveness and sensitivity to both acute and chronic stress exposure. Children with chronic health conditions, such as asthma or obesity, may exhibit dysregulation of the HPA axis, which has implications for both physical and psychological health outcomes. Despite the growing recognition of psychoneuroendocrine interactions, the role of structured environmental interventions—such as spa treatment—in modulating stress biomarkers in children remains underexplored. The aim of this study was to evaluate the impact of a standardized 28-day spa treatment on salivary cortisol levels in children aged 9–12 years. Salivary cortisol served as a surrogate biomarker of stress regulation via the HPA axis. We hypothesized that spa interventions would lead to measurable changes in cortisol levels, reflective of diagnosis-specific neuroendocrine responses. A secondary objective was to assess the utility of salivary cortisol as a stress-sensitive biomarker within pediatric rehabilitation contexts. changes in salivary cortisol levels in children with obesity and chronic respiratory diseases after 28 days of treatment, differences between diagnostic groups and an age-matched control group of healthy children, the extent of the hormonal response to physical and lifestyle input depending on the underlying diagnosis. A secondary aim was to assess the suitability of salivary cortisol as a biomarker of physiological adaptation in the context of comprehensive spa therapy. Methods 1. Study Design and General Information The study was designed as a longitudinal, non-randomized, prospective observational study evaluating the effects of spa treatment on selected health parameters in children aged 9–12 years. The research was conducted in 2022 and followed pilot studies carried out in 2020 and 2021. Children were admitted to three geographically distinct pediatric spa facilities in the Czech Republic, designated in the statistical analysis as Groups 1, 2, and 3. Assessments were conducted within the first days after admission and again after at least 21 days (range: 21–26 days). The control group consisted of 38 children from a rural primary school, who underwent repeated measurements 24 days apart. The primary objective of this study was to evaluate the impact of spa treatment on the neuroendocrine response in children aged 9–12 years through changes in salivary cortisol levels as a non-invasive marker of HPA axis activity and to compare these values with somatic data and functional test results. 2. Sample Selection and Characteristics Out of 440 initially screened candidates, 95 children (45% boys, 55% girls) were enrolled after applying strict exclusion criteria, which included: Age outside the 9–12 year range, Signs of neurological pathology at baseline examination (e.g., balance disorders, positive Romberg test, nystagmus), Chronic use of hormone replacement therapy (e.g., corticosteroids in any form, hypothyroidism treatment), Presence of menarche in girls, BMI below 12, Complications during treatment (e.g., injuries, infections). The control group had a similar age distribution (mean age: 10.22 years) and sex ratio (47% boys, 53% girls). All participants exhibited normal respiratory rates (20–30 breaths per minute). 3. Diagnostic Classification and Salivary Cortisol Sampling Among the 95 children undergoing spa treatment: 50 had chronic respiratory diseases (mainly bronchial asthma), 36 were classified as obese, 5 had urinary tract disorders, 4 were treated for atopic eczema. Salivary cortisol was collected in the morning between 7:45 and 8:30 a.m., corresponding to the expected circadian peak. Sterile Salivette swabs were used, and samples were frozen and transported to the Institute of Endocrinology in Prague for analysis. In several cases, sampling was unsuccessful due to technical issues (e.g., insufficient saliva production). 4. Testing Methods The test battery included clinically established, age-appropriate screening procedures selected to minimize stress and accommodate the psychomotor development of 9–12-year-old children. Assessments included standard somatometric data (height, weight, limb circumference), peripheral oxygen saturation, resting heart rate, chest circumference (inspiration/expiration), waist and hip circumference, BMI calculation, and body composition analysis using bioimpedance (TANITA BC-100, Health Monitor GMON software). Pulmonary function was assessed via spirometry (vital capacity [VC] and forced expiratory volume in 1 second [FEV₁]) using a portable ultrasonic spirometer (SpiroSonic FLO, SpiroReporter software), following updated ATS/ERS 2019 guidelines [ 5 ]. Salivary cortisol was measured in accordance with standardized protocols of the Institute of Endocrinology in Prague using validated assay kits [ 6 ]. Physical fitness was assessed using the 6-Minute Walk Test (6MWT), based on recommendations of the professional pulmonology society [ 7 ]. Motor coordination and agility were assessed using the Four Square Step Test (FSST), a standard tool for evaluating dynamic balance [ 8 ]. Statistical Analysis Raw metric data were transformed for parametric testing using power transformations to achieve symmetry and homoscedasticity [ 9 ]. Relationships among the outcome variables, treatment location, and study phase were evaluated using a linear model, which included the Subject factor (inter-individual variability), fixed factor Location (no repetition), repeated-measures factor Phase (study phase), interaction Location × Phase, and the covariate of saliva sampling time (to account for circadian rhythm). Symmetry, stability, and homogeneity of data distributions and residuals were verified using regression diagnostics [ 10 , 11 ]. Following linear modeling, multiple comparisons were performed using the least significant difference (LSD) method. Relationships among changes in cortisol levels, study location, anthropometric indicators, and changes in anthropometric variables were further analyzed using multiple regression with dimensionality reduction via orthogonal projections to latent structures (OPLS) [ 12 ], due to high multicollinearity among explanatory variables. Specific associations between the dependent and independent variables (adjusted for covariates) were evaluated using standard multiple regression. Sample Characteristics In 2022, a total of 133 children aged 9–12 years were included in the study. Of these, 95 children were enrolled as the experimental cohort undergoing spa treatment at three geographically distinct pediatric spa centers (Groups 1–3). Participants were recruited from across the Czech Republic. The control group consisted of 38 randomly selected healthy children from a rural primary school. Entry and exit measurements were conducted 21–26 days apart in the treatment group and precisely 24 days apart in the control group. The treatment group included 43 boys (45.3%) and 52 girls (54.7%); the control group included 18 boys (47.4%) and 20 girls (52.6%). The mean age in the treatment group was 10.87 years, and 10.22 years in the control group. Age and sex distributions were comparable, although the control group was slightly younger on average, which was clinically insignificant. Only 6 children in the treatment group (6.3%) were left-handed; none in the control group were. Diagnostic breakdown of the treatment group (n = 95): Obesity: 36 children (37.9%), Chronic respiratory diseases: 50 children (52.6%), Nephrourological conditions: 5 children, Atopic eczema: 4 children. The largest subgroup consisted of children with respiratory diseases, most commonly bronchial asthma. A total of 33 children were excluded due to ongoing corticosteroid therapy. Salivary cortisol was successfully measured at both time points in: 34 of 36 children with obesity, 18 of 50 children with respiratory conditions, 6 of 9 children with other diagnoses (atopic eczema n = 4; bladder dysfunction n = 5), 36 of 38 children in the control group. Ethical Considerations The study was approved by the Ethics Committee of the Institute of Endocrinology in Prague. Written informed consent was obtained from all participants’ legal guardians and from the children themselves prior to study enrollment. The study was conducted in accordance with the Declaration of Helsinki. Results 1. Anthropometric and Functional Parameters In the spa treatment group, the average BMI decreased by 0.7 points, with the most notable reductions observed in obese children. The Six-Minute Walk Test (6MWT) showed a 12% increase in the average walking distance, indicating an improvement in physical fitness. These changes were not statistically significant in the control group. 2. Kinesiological and Balance Tests Kinesiological assessment using the Computer Kinesiology system revealed improved functional status of the musculoskeletal system in 68% of children in the spa group. The Four Square Step Test (FSST) demonstrated enhanced dynamic balance and agility in 54% of these children. No significant changes were observed in these tests within the control group. 3. Correlations Between Parameters Analysis using the orthogonal projections to latent structures (OPLS) method revealed a strong correlation between the reduction in salivary cortisol levels and improvements in physical fitness (6MWT) and BMI reduction. This correlation was most pronounced in the subgroup of obese children. 4. Changes in Salivary Cortisol Levels Morning salivary cortisol levels were assessed in children aged 9–12 years at the beginning (IN) and end (OUT) of spa treatment. Results revealed diagnosis-dependent trends in cortisol changes, with details and statistical significance visualized in Fig. 1: Children with obesity showed a decrease in mean cortisol levels from 3.32 nmol/l to 2.36 nmol/l. Children with respiratory diseases (primarily bronchial asthma and post-infectious conditions of the lower respiratory tract) exhibited an increase from 2.68 nmol/l to 3.48 nmol/l. Children with atopic eczema showed a slight increase from 3.08 to 3.60 nmol/l. Children with nephrourological diagnoses had higher baseline values (5.48 nmol/l), with a mild decrease to 5.28 nmol/l. Healthy children in the control group had an initial mean cortisol level of 3.52 nmol/l, which increased to 4.02 nmol/l at follow-up. The results are illustrated in Fig. 1, which displays salivary cortisol values across diagnostic groups. Figure 1. Salivary cortisol levels by diagnostic group Dgmain: F = 13.8, p < 0.001, ηp2 = 0.372; Stage: F = 3.5, p = 0.066, ηp2 = 0.0358; GDM × Stage: F = 7.7, p < 0.001, ηp2 = 0.25; ; Subj(GDM): F = 4.3, p HC, OB < HC, RD < BD, DE < BD, OB < BD, OB HC1, OB2 < OB1 Legend: HC – healthy controls; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group; Stage represents testing at baseline (1) and post-treatment (2). To provide a more comprehensive understanding of the relationships between salivary cortisol levels and other measured parameters, the results of the O2PLS model are included in the supplementary materials—specifically for the subgroup of patients with obesity (Appendix 1) and the subgroup with respiratory diseases (Appendix 2). The intercorrelation analysis demonstrates distinct response patterns between these patient groups. Figure 2 presents the primary classification of children by BMI in relation to their primary diagnosis. Figure 2. Body mass index (BMI) of individual participant groups according to primary diagnosis, with annotations of statistically significant changes. Dgmain: F = 9047.1, p < 0.001, ηp2 = 0.997; Stage: F = 75.4, p < 0.001, ηp2 = 0.382; GDM × Stage: F = 51, p < 0.001, ηp2 = 0.626; ; Subj(GDM): F = 264, p < 0.001, ηp2 = 0.996; BD HC, DE > HC, OB > HC, RD > BD, DE > BD, OB > BD, DE > RD, OB > RD, OB > DE, HC2 > HC1, BD2 < BD1, RD2 < RD1, OB2 < OB1 Legend: HC – control group; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2). A specific subgroup of children with chronic respiratory diseases (primarily bronchial asthma) was also identified, in whom neither obesity nor corticosteroid therapy was present. This resulted in a comparable cohort of children with a homogeneous diagnosis and without major confounding factors. Discussion The salivary cortisol measurements confirmed a downward trend in cortisol levels among children with obesity. Physical exercise combined with dietary regulation and weight reduction led to a statistically significant decrease in salivary cortisol levels, suggesting a favorable hormonal adaptation. As a key hormone regulating carbohydrate, lipid, and protein metabolism, salivary cortisol may serve as a marker of physiological reactivity to therapeutic load within the framework of comprehensive spa rehabilitative care (CSRRC). We believe that the observed cortisol reduction in obese children reflects decreased stress levels in response to a healthier physical and lifestyle pattern—and that similar results could potentially be achieved in a home setting with family support. In contrast, children with chronic respiratory conditions, primarily asthma, demonstrated increased cortisol levels after treatment. This pattern may reflect the reactivation of an underresponsive HPA axis often observed in children with chronic inflammatory diseases, who may exhibit blunted stress responses at baseline. The rise in cortisol post-intervention may indicate a shift toward more dynamic stress system responsiveness—a potential marker of physiological adaptation rather than dysregulation. These findings align with psychoneuroimmunological models that describe altered HPA axis function in chronic disease and its modulation through environmental and behavioral inputs. Establishing reference values for salivary cortisol, both in healthy populations and in children with chronic diseases, remains challenging. As an example of published normative data, Safarzadeh et al. [ 13 ] reported salivary cortisol levels ranging from 1.7 to 12.8 nmol/l. These findings are supported by parallel improvements in physical function and neuromotor integration, as observed in the Six-Minute Walk Test (6MWT) and the Four Square Step Test (FSST). These objective measures of mobility and coordination further illustrate the complex systemic adaptations to structured rehabilitation settings. Importantly, the distinct cortisol trajectories between diagnostic groups underscore the relevance of salivary cortisol as a nuanced biomarker of stress regulation and physiological plasticity in pediatric populations. Future studies should consider complementing salivary cortisol data with additional stress markers and immune parameters to better delineate the biopsychosocial mechanisms involved. The results of the Six-Minute Walk Test are shown in Fig. 3. Figure 3. Summary results of the Six-Minute Walk Test (6MWT) Dgmain: F = 83.5, p < 0.001, ηp2 = 0.738; Stage: F = 11.7, p = 0.001, ηp2 = 0.116; GDM × Stage: F = 1.2, p = 0.312, ηp2 = 0.0391; Subj(GDM): F = 3.5, p < 0.001, ηp2 = 0.782; DE < BD, OB < BD, OB < RD, OB < DE, Legend: BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2). Figure 4. Summary results of individual participant groups in the Four Square Step Test (FSST) Dgmain: F = 1.1, p = 0.384, ηp2 = 0.0341; Stage: F = 36.8, p < 0.001, ηp2 = 0.236; GDM × Stage: F = 3.2, p = 0.015, ηp2 = 0.098; ; Subj(GDM): F = 6.4, p < 0.001, ηp2 = 0.867; RD2 < RD1, OB2 < OB1 Legend: HC – control group; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2). Conclusion This pilot prospective study demonstrated that a 28-day spa treatment leads to measurable changes in salivary cortisol levels in children aged 9–12 years with obesity and respiratory diseases. In obese children, a statistically significant decrease in cortisol was observed, which may be interpreted as a favorable hormonal response to weight reduction, likely associated with decreased production of proinflammatory mediators by adipose tissue. Weight loss appears to contribute to improved emotional well-being and to a broader enhancement of limbic system function, with downstream effects on key regulatory axes (psyche, immunity, metabolism). In contrast, an increase in cortisol levels was observed in children with chronic respiratory diseases, possibly reflecting heightened HPA axis activation triggered by structured physical activity and routine interventions that include adequate sleep. Agility test results (FSST) showed a statistically significant improvement in children with respiratory diseases and those who were overweight or obese (Fig. 4). Comparable improvements were observed in the two remaining diagnostic groups; however, due to the small number of participants in these subgroups, the results could not be statistically confirmed. The Six-Minute Walk Test (6MWT) results demonstrated a stratification of physical fitness according to diagnostic group. Limitations Given the pilot nature of this study and the limited size of some subgroups, the findings should be interpreted with caution. Nevertheless, the study paves the way for further research into the relationships between body composition, hormonal regulation, and therapeutic response within the context of pediatric spa treatment. Clinical Implications Salivary cortisol can be used as a sensitive, non-invasive, and repeatable marker of neuroendocrine activity in children undergoing inpatient treatment. In children with obesity, a decrease in cortisol may indicate not only a reduction in stress burden but also a favorable metabolic response to changes in physical activity and lifestyle. An increase in cortisol among children with respiratory diseases may reflect reactivation of the HPA axis following prior pharmacotherapy or increased physical exertion and should be interpreted in the context of the individual clinical condition. These findings support the use of spa treatment as a comprehensive therapeutic environment that positively influences the health status of children with chronic illnesses. Abbreviations BMI – Body Mass Index CSRRC – Comprehensive Spa Rehabilitative Care DE – Dermatological Indication Group FFM – Fat-Free Mass FSST – Four Square Step Test HPA axis – Hypothalamic–Pituitary–Adrenal Axis IN/OUT – Initial and Final Measurement Points OB – Obesity Indication Group OPLS/O2PLS – Orthogonal Projections to Latent Structures / Bidirectional O2PLS RD – Respiratory Disease Indication Group SpO₂ – Peripheral Oxygen Saturation 6MWT – Six-Minute Walk Test PNIE axis – Psycho–Neuro–Immune–Endocrine Axis VC/FEV₁ – Vital Capacity / Forced Expiratory Volume in 1 Second HC – Healthy Controls (Control Group) Declarations Ethics approval and consent to participate The study was performed in line with the principles of the Declaration of Helsinki. Approval was obtained from the Ethics Committee of the Institute of Endocrinology (Prague, Czech Republic). Written informed consent was obtained from all parents or legal guardians of the participating children prior to enrollment. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was supported by MH CZ – DRO (Institute of Endocrinology – EÚ, 00023761). Author Contribution Z.T. conceived the study, coordinated data collection, and drafted the main manuscript text.D.J. contributed to clinical data acquisition and patient management.M.H. performed statistical analyses and contributed to data interpretation.M.B. and L.M. conducted hormonal analyses of salivary cortisol and supervised laboratory procedures.L.M. also assisted with literature review, data organization, and manuscript editing.All authors reviewed and approved the final version of the manuscript. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in European Journal of Pediatrics → Version 1 posted Editorial decision: Revision requested 16 Oct, 2025 Reviews received at journal 16 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviews received at journal 01 Oct, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers invited by journal 09 Sep, 2025 Editor assigned by journal 08 Sep, 2025 Submission checks completed at journal 08 Sep, 2025 First submitted to journal 28 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7481334","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515774608,"identity":"1a6b6d67-b4c6-4eb5-8ef3-446877752196","order_by":0,"name":"Zdeněk 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06:46:06","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63263,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/d20144c294f378d89430200e.html"},{"id":91815959,"identity":"c489d70b-aa2c-4875-95c0-b4e890ab7f3d","added_by":"auto","created_at":"2025-09-22 06:38:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83702,"visible":true,"origin":"","legend":"\u003cp\u003eSalivary cortisol levels by diagnostic group\u003c/p\u003e\n\u003cp\u003eDgmain: F=13.8, p\u0026lt;0.001, ηp2=0.372; Stage: F=3.5, p=0.066, ηp2=0.0358; GDM × Stage: F=7.7, p\u0026lt;0.001, ηp2=0.25; ; Subj(GDM): F=4.3, p\u0026lt;0.001, ηp2=0.815; BD\u0026gt;HC, OB\u0026lt;HC, RD\u0026lt;BD, DE\u0026lt;BD, OB\u0026lt;BD, OB\u0026lt;RD, HC2\u0026gt;HC1, OB2\u0026lt;OB1\u003c/p\u003e\n\u003cp\u003eLegend: HC – healthy controls; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group; Stage represents testing at baseline (1) and post-treatment (2).\u003c/p\u003e\n\u003cp\u003eTo provide a more comprehensive understanding of the relationships between salivary cortisol levels and other measured parameters, the results of the O2PLS model are included in the supplementary materials—specifically for the subgroup of patients with obesity (Appendix 1) and the subgroup with respiratory diseases (Appendix 2). The intercorrelation analysis demonstrates distinct response patterns between these patient groups. Figure 2 presents the primary classification of children by BMI in relation to their primary diagnosis.\u003c/p\u003e","description":"","filename":"Triskalafig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/89b430ae6e27ab11dafb9004.jpg"},{"id":91815674,"identity":"5eb4aa38-a3c9-4592-b136-96f048ed7010","added_by":"auto","created_at":"2025-09-22 06:30:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99863,"visible":true,"origin":"","legend":"\u003cp\u003eBody mass index (BMI) of individual participant groups according to primary diagnosis, with annotations of statistically significant changes.\u003c/p\u003e\n\u003cp\u003eDgmain: F=9047.1, p\u0026lt;0.001, ηp2=0.997; Stage: F=75.4, p\u0026lt;0.001, ηp2=0.382; GDM × Stage: F=51, p\u0026lt;0.001, ηp2=0.626; ; Subj(GDM): F=264, p\u0026lt;0.001, ηp2=0.996; BD\u0026lt;HC, RD\u0026gt;HC, DE\u0026gt;HC, OB\u0026gt;HC, RD\u0026gt;BD, DE\u0026gt;BD, OB\u0026gt;BD, DE\u0026gt;RD, OB\u0026gt;RD, OB\u0026gt;DE, HC2\u0026gt;HC1, BD2\u0026lt;BD1, RD2\u0026lt;RD1, OB2\u0026lt;OB1\u003c/p\u003e\n\u003cp\u003eLegend: HC – control group; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e","description":"","filename":"Triskalafig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/035be17aedb6e633e7037f6f.jpg"},{"id":91816554,"identity":"faf1adb5-8bee-4a92-bee9-92b5a8d10a1b","added_by":"auto","created_at":"2025-09-22 06:46:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79356,"visible":true,"origin":"","legend":"\u003cp\u003eSummary results of the Six-Minute Walk Test (6MWT)\u003c/p\u003e\n\u003cp\u003eDgmain: F=83.5, p\u0026lt;0.001, ηp2=0.738; Stage: F=11.7, p=0.001, ηp2=0.116; GDM × Stage: F=1.2, p=0.312, ηp2=0.0391; Subj(GDM): F=3.5, p\u0026lt;0.001, ηp2=0.782; DE\u0026lt;BD, OB\u0026lt;BD, OB\u0026lt;RD, OB\u0026lt;DE,\u003c/p\u003e\n\u003cp\u003eLegend: BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e","description":"","filename":"Triskalafig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/935391ef97b57c7bb82bde52.jpg"},{"id":91815963,"identity":"661a25f3-7a42-44ef-8e3b-26569b6d8994","added_by":"auto","created_at":"2025-09-22 06:38:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90944,"visible":true,"origin":"","legend":"\u003cp\u003eSummary results of individual participant groups in the Four Square Step Test (FSST)\u003c/p\u003e\n\u003cp\u003eDgmain: F=1.1, p=0.384, ηp2=0.0341; Stage: F=36.8, p\u0026lt;0.001, ηp2=0.236; GDM × Stage: F=3.2, p=0.015, ηp2=0.098; ; Subj(GDM): F=6.4, p\u0026lt;0.001, ηp2=0.867; RD2\u0026lt;RD1, OB2\u0026lt;OB1\u003c/p\u003e\n\u003cp\u003eLegend: HC – control group; BD – nephrological indication group; RD – respiratory disease indication group; DE – dermatological indication group; OB – obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e","description":"","filename":"Triskalafig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/9cc373b89ba23938c939babe.jpg"},{"id":100614767,"identity":"925c23b6-021c-4db9-acd1-bc3d939e7e56","added_by":"auto","created_at":"2026-01-19 17:24:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":937175,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7481334/v1/bb2bdc5f-f46b-491d-9aa5-db217ee622af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Change in Salivary Cortisol Levels in Children (Aged 9–12 Years) with Obesity and Respiratory Diseases during a 28-Day Spa Treatment: A Pilot Prospective Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent decades, there has been growing interest in the objective assessment of spa treatment effects not only on somatic health but also on neuroendocrine and psychosocial functions. This approach is grounded in the concept of the psycho\u0026ndash;neuro\u0026ndash;immune\u0026ndash;endocrine (PNIE) axis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which highlights the interconnectedness of the central nervous system, endocrine regulation, immune responses, and an individual\u0026rsquo;s psychological state [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This integrative perspective is well captured by the assertion: \u0026ldquo;The musculoskeletal system responds to all external and internal dysfunctions of the body in an integrative manner, and most of them can be influenced or eliminated through the musculoskeletal system.\u0026rdquo; [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Spa treatment, which combines physical activity, dietary regulation, and exposure to stimulating natural environments, provides a natural framework for studying complex adaptive mechanisms.\u003c/p\u003e\u003cp\u003ePrevious studies in adult populations undergoing structured spa or rehabilitation treatments have shown that such interventions may modulate stress physiology, leading to reduced cortisol levels and improved neuroendocrine regulation. These effects are often accompanied by enhancements in subjective well-being, physical fitness, and metabolic health. Within this context, salivary cortisol\u0026mdash;an established non-invasive biomarker of hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis activation\u0026mdash;provides an accessible index of stress-related physiological states.\u003c/p\u003e\u003cp\u003eIn pediatric populations, the measurement of salivary cortisol is particularly advantageous due to its non-invasiveness and sensitivity to both acute and chronic stress exposure. Children with chronic health conditions, such as asthma or obesity, may exhibit dysregulation of the HPA axis, which has implications for both physical and psychological health outcomes. Despite the growing recognition of psychoneuroendocrine interactions, the role of structured environmental interventions\u0026mdash;such as spa treatment\u0026mdash;in modulating stress biomarkers in children remains underexplored.\u003c/p\u003e\u003cp\u003eThe aim of this study was to evaluate the impact of a standardized 28-day spa treatment on salivary cortisol levels in children aged 9\u0026ndash;12 years. Salivary cortisol served as a surrogate biomarker of stress regulation via the HPA axis. We hypothesized that spa interventions would lead to measurable changes in cortisol levels, reflective of diagnosis-specific neuroendocrine responses. A secondary objective was to assess the utility of salivary cortisol as a stress-sensitive biomarker within pediatric rehabilitation contexts.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003echanges in salivary cortisol levels in children with obesity and chronic respiratory diseases after 28 days of treatment,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003edifferences between diagnostic groups and an age-matched control group of healthy children,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ethe extent of the hormonal response to physical and lifestyle input depending on the underlying diagnosis.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eA secondary aim was to assess the suitability of salivary cortisol as a biomarker of physiological adaptation in the context of comprehensive spa therapy.\u003c/p\u003e"},{"header":"Methods","content":"\n\u003ch3\u003e1. Study Design and General Information\u003c/h3\u003e\n\u003cp\u003eThe study was designed as a longitudinal, non-randomized, prospective observational study evaluating the effects of spa treatment on selected health parameters in children aged 9\u0026ndash;12 years. The research was conducted in 2022 and followed pilot studies carried out in 2020 and 2021. Children were admitted to three geographically distinct pediatric spa facilities in the Czech Republic, designated in the statistical analysis as Groups 1, 2, and 3. Assessments were conducted within the first days after admission and again after at least 21 days (range: 21\u0026ndash;26 days).\u003c/p\u003e\u003cp\u003eThe control group consisted of 38 children from a rural primary school, who underwent repeated measurements 24 days apart.\u003c/p\u003e\u003cp\u003eThe primary objective of this study was to evaluate the impact of spa treatment on the neuroendocrine response in children aged 9\u0026ndash;12 years through changes in salivary cortisol levels as a non-invasive marker of HPA axis activity and to compare these values with somatic data and functional test results.\u003c/p\u003e\n\u003ch3\u003e2. Sample Selection and Characteristics\u003c/h3\u003e\n\u003cp\u003eOut of 440 initially screened candidates, 95 children (45% boys, 55% girls) were enrolled after applying strict exclusion criteria, which included:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eAge outside the 9\u0026ndash;12 year range,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eSigns of neurological pathology at baseline examination (e.g., balance disorders, positive Romberg test, nystagmus),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChronic use of hormone replacement therapy (e.g., corticosteroids in any form, hypothyroidism treatment),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePresence of menarche in girls,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBMI below 12,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eComplications during treatment (e.g., injuries, infections).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe control group had a similar age distribution (mean age: 10.22 years) and sex ratio (47% boys, 53% girls). All participants exhibited normal respiratory rates (20\u0026ndash;30 breaths per minute).\u003c/p\u003e\n\u003ch3\u003e3. Diagnostic Classification and Salivary Cortisol Sampling\u003c/h3\u003e\n\u003cp\u003eAmong the 95 children undergoing spa treatment:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e50 had chronic respiratory diseases (mainly bronchial asthma),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e36 were classified as obese,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e5 had urinary tract disorders,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e4 were treated for atopic eczema.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eSalivary cortisol was collected in the morning between 7:45 and 8:30 a.m., corresponding to the expected circadian peak. Sterile Salivette swabs were used, and samples were frozen and transported to the Institute of Endocrinology in Prague for analysis. In several cases, sampling was unsuccessful due to technical issues (e.g., insufficient saliva production).\u003c/p\u003e\n\u003ch3\u003e4. Testing Methods\u003c/h3\u003e\n\u003cp\u003eThe test battery included clinically established, age-appropriate screening procedures selected to minimize stress and accommodate the psychomotor development of 9\u0026ndash;12-year-old children. Assessments included standard somatometric data (height, weight, limb circumference), peripheral oxygen saturation, resting heart rate, chest circumference (inspiration/expiration), waist and hip circumference, BMI calculation, and body composition analysis using bioimpedance (TANITA BC-100, Health Monitor GMON software).\u003c/p\u003e\u003cp\u003ePulmonary function was assessed via spirometry (vital capacity [VC] and forced expiratory volume in 1 second [FEV₁]) using a portable ultrasonic spirometer (SpiroSonic FLO, SpiroReporter software), following updated ATS/ERS 2019 guidelines [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSalivary cortisol was measured in accordance with standardized protocols of the Institute of Endocrinology in Prague using validated assay kits [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePhysical fitness was assessed using the 6-Minute Walk Test (6MWT), based on recommendations of the professional pulmonology society [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMotor coordination and agility were assessed using the Four Square Step Test (FSST), a standard tool for evaluating dynamic balance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eRaw metric data were transformed for parametric testing using power transformations to achieve symmetry and homoscedasticity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Relationships among the outcome variables, treatment location, and study phase were evaluated using a linear model, which included the Subject factor (inter-individual variability), fixed factor Location (no repetition), repeated-measures factor Phase (study phase), interaction Location \u0026times; Phase, and the covariate of saliva sampling time (to account for circadian rhythm). Symmetry, stability, and homogeneity of data distributions and residuals were verified using regression diagnostics [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFollowing linear modeling, multiple comparisons were performed using the least significant difference (LSD) method. Relationships among changes in cortisol levels, study location, anthropometric indicators, and changes in anthropometric variables were further analyzed using multiple regression with dimensionality reduction via orthogonal projections to latent structures (OPLS) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], due to high multicollinearity among explanatory variables. Specific associations between the dependent and independent variables (adjusted for covariates) were evaluated using standard multiple regression.\u003c/p\u003e\u003cp\u003eSample Characteristics\u003c/p\u003e\u003cp\u003eIn 2022, a total of 133 children aged 9\u0026ndash;12 years were included in the study. Of these, 95 children were enrolled as the experimental cohort undergoing spa treatment at three geographically distinct pediatric spa centers (Groups 1\u0026ndash;3). Participants were recruited from across the Czech Republic.\u003c/p\u003e\u003cp\u003eThe control group consisted of 38 randomly selected healthy children from a rural primary school. Entry and exit measurements were conducted 21\u0026ndash;26 days apart in the treatment group and precisely 24 days apart in the control group.\u003c/p\u003e\u003cp\u003eThe treatment group included 43 boys (45.3%) and 52 girls (54.7%); the control group included 18 boys (47.4%) and 20 girls (52.6%). The mean age in the treatment group was 10.87 years, and 10.22 years in the control group. Age and sex distributions were comparable, although the control group was slightly younger on average, which was clinically insignificant. Only 6 children in the treatment group (6.3%) were left-handed; none in the control group were.\u003c/p\u003e\u003cp\u003eDiagnostic breakdown of the treatment group (n\u0026thinsp;=\u0026thinsp;95):\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eObesity: 36 children (37.9%),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChronic respiratory diseases: 50 children (52.6%),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eNephrourological conditions: 5 children,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAtopic eczema: 4 children.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe largest subgroup consisted of children with respiratory diseases, most commonly bronchial asthma.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eA total of 33 children were excluded due to ongoing corticosteroid therapy. Salivary cortisol was successfully measured at both time points in:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e34 of 36 children with obesity,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e18 of 50 children with respiratory conditions,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e6 of 9 children with other diagnoses (atopic eczema n\u0026thinsp;=\u0026thinsp;4; bladder dysfunction n\u0026thinsp;=\u0026thinsp;5),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e36 of 38 children in the control group.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eEthical Considerations\u003c/p\u003e\u003cp\u003e The study was approved by the Ethics Committee of the Institute of Endocrinology in Prague. Written informed consent was obtained from all participants\u0026rsquo; legal guardians and from the children themselves prior to study enrollment. The study was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1. Anthropometric and Functional Parameters\u003c/h3\u003e\n\u003cp\u003eIn the spa treatment group, the average BMI decreased by 0.7 points, with the most notable reductions observed in obese children. The Six-Minute Walk Test (6MWT) showed a 12% increase in the average walking distance, indicating an improvement in physical fitness. These changes were not statistically significant in the control group.\u003c/p\u003e\n\u003ch3\u003e2. Kinesiological and Balance Tests\u003c/h3\u003e\n\u003cp\u003eKinesiological assessment using the Computer Kinesiology system revealed improved functional status of the musculoskeletal system in 68% of children in the spa group. The Four Square Step Test (FSST) demonstrated enhanced dynamic balance and agility in 54% of these children. No significant changes were observed in these tests within the control group.\u003c/p\u003e\n\u003ch3\u003e3. Correlations Between Parameters\u003c/h3\u003e\n\u003cp\u003eAnalysis using the orthogonal projections to latent structures (OPLS) method revealed a strong correlation between the reduction in salivary cortisol levels and improvements in physical fitness (6MWT) and BMI reduction. This correlation was most pronounced in the subgroup of obese children.\u003c/p\u003e\n\u003ch3\u003e4. Changes in Salivary Cortisol Levels\u003c/h3\u003e\n\u003cp\u003eMorning salivary cortisol levels were assessed in children aged 9\u0026ndash;12 years at the beginning (IN) and end (OUT) of spa treatment. Results revealed diagnosis-dependent trends in cortisol changes, with details and statistical significance visualized in Fig.\u0026nbsp;1:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eChildren with obesity showed a decrease in mean cortisol levels from 3.32 nmol/l to 2.36 nmol/l.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChildren with respiratory diseases (primarily bronchial asthma and post-infectious conditions of the lower respiratory tract) exhibited an increase from 2.68 nmol/l to 3.48 nmol/l.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChildren with atopic eczema showed a slight increase from 3.08 to 3.60 nmol/l.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChildren with nephrourological diagnoses had higher baseline values (5.48 nmol/l), with a mild decrease to 5.28 nmol/l.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHealthy children in the control group had an initial mean cortisol level of 3.52 nmol/l, which increased to 4.02 nmol/l at follow-up.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe results are illustrated in Fig.\u0026nbsp;1, which displays salivary cortisol values across diagnostic groups.\u003c/p\u003e\u003cp\u003eFigure 1. Salivary cortisol levels by diagnostic group\u003c/p\u003e\u003cp\u003eDgmain: F\u0026thinsp;=\u0026thinsp;13.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.372; Stage: F\u0026thinsp;=\u0026thinsp;3.5, p\u0026thinsp;=\u0026thinsp;0.066, ηp2\u0026thinsp;=\u0026thinsp;0.0358; GDM \u0026times; Stage: F\u0026thinsp;=\u0026thinsp;7.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.25; ; Subj(GDM): F\u0026thinsp;=\u0026thinsp;4.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.815; BD\u0026thinsp;\u0026gt;\u0026thinsp;HC, OB\u0026thinsp;\u0026lt;\u0026thinsp;HC, RD\u0026thinsp;\u0026lt;\u0026thinsp;BD, DE\u0026thinsp;\u0026lt;\u0026thinsp;BD, OB\u0026thinsp;\u0026lt;\u0026thinsp;BD, OB\u0026thinsp;\u0026lt;\u0026thinsp;RD, HC2\u0026thinsp;\u0026gt;\u0026thinsp;HC1, OB2\u0026thinsp;\u0026lt;\u0026thinsp;OB1\u003c/p\u003e\u003cp\u003eLegend: HC \u0026ndash; healthy controls; BD \u0026ndash; nephrological indication group; RD \u0026ndash; respiratory disease indication group; DE \u0026ndash; dermatological indication group; OB \u0026ndash; obesity indication group; Stage represents testing at baseline (1) and post-treatment (2).\u003c/p\u003e\u003cp\u003eTo provide a more comprehensive understanding of the relationships between salivary cortisol levels and other measured parameters, the results of the O2PLS model are included in the supplementary materials\u0026mdash;specifically for the subgroup of patients with obesity (Appendix 1) and the subgroup with respiratory diseases (Appendix 2). The intercorrelation analysis demonstrates distinct response patterns between these patient groups. Figure\u0026nbsp;2 presents the primary classification of children by BMI in relation to their primary diagnosis.\u003c/p\u003e\u003cp\u003eFigure 2. Body mass index (BMI) of individual participant groups according to primary diagnosis, with annotations of statistically significant changes.\u003c/p\u003e\u003cp\u003eDgmain: F\u0026thinsp;=\u0026thinsp;9047.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.997; Stage: F\u0026thinsp;=\u0026thinsp;75.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.382; GDM \u0026times; Stage: F\u0026thinsp;=\u0026thinsp;51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.626; ; Subj(GDM): F\u0026thinsp;=\u0026thinsp;264, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.996; BD\u0026thinsp;\u0026lt;\u0026thinsp;HC, RD\u0026thinsp;\u0026gt;\u0026thinsp;HC, DE\u0026thinsp;\u0026gt;\u0026thinsp;HC, OB\u0026thinsp;\u0026gt;\u0026thinsp;HC, RD\u0026thinsp;\u0026gt;\u0026thinsp;BD, DE\u0026thinsp;\u0026gt;\u0026thinsp;BD, OB\u0026thinsp;\u0026gt;\u0026thinsp;BD, DE\u0026thinsp;\u0026gt;\u0026thinsp;RD, OB\u0026thinsp;\u0026gt;\u0026thinsp;RD, OB\u0026thinsp;\u0026gt;\u0026thinsp;DE, HC2\u0026thinsp;\u0026gt;\u0026thinsp;HC1, BD2\u0026thinsp;\u0026lt;\u0026thinsp;BD1, RD2\u0026thinsp;\u0026lt;\u0026thinsp;RD1, OB2\u0026thinsp;\u0026lt;\u0026thinsp;OB1\u003c/p\u003e\u003cp\u003eLegend: HC \u0026ndash; control group; BD \u0026ndash; nephrological indication group; RD \u0026ndash; respiratory disease indication group; DE \u0026ndash; dermatological indication group; OB \u0026ndash; obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e\u003cp\u003eA specific subgroup of children with chronic respiratory diseases (primarily bronchial asthma) was also identified, in whom neither obesity nor corticosteroid therapy was present. This resulted in a comparable cohort of children with a homogeneous diagnosis and without major confounding factors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe salivary cortisol measurements confirmed a downward trend in cortisol levels among children with obesity. Physical exercise combined with dietary regulation and weight reduction led to a statistically significant decrease in salivary cortisol levels, suggesting a favorable hormonal adaptation. As a key hormone regulating carbohydrate, lipid, and protein metabolism, salivary cortisol may serve as a marker of physiological reactivity to therapeutic load within the framework of comprehensive spa rehabilitative care (CSRRC). We believe that the observed cortisol reduction in obese children reflects decreased stress levels in response to a healthier physical and lifestyle pattern\u0026mdash;and that similar results could potentially be achieved in a home setting with family support.\u003c/p\u003e\u003cp\u003eIn contrast, children with chronic respiratory conditions, primarily asthma, demonstrated increased cortisol levels after treatment. This pattern may reflect the reactivation of an underresponsive HPA axis often observed in children with chronic inflammatory diseases, who may exhibit blunted stress responses at baseline. The rise in cortisol post-intervention may indicate a shift toward more dynamic stress system responsiveness\u0026mdash;a potential marker of physiological adaptation rather than dysregulation. These findings align with psychoneuroimmunological models that describe altered HPA axis function in chronic disease and its modulation through environmental and behavioral inputs.\u003c/p\u003e\u003cp\u003eEstablishing reference values for salivary cortisol, both in healthy populations and in children with chronic diseases, remains challenging. As an example of published normative data, Safarzadeh et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] reported salivary cortisol levels ranging from 1.7 to 12.8 nmol/l.\u003c/p\u003e\u003cp\u003eThese findings are supported by parallel improvements in physical function and neuromotor integration, as observed in the Six-Minute Walk Test (6MWT) and the Four Square Step Test (FSST). These objective measures of mobility and coordination further illustrate the complex systemic adaptations to structured rehabilitation settings. Importantly, the distinct cortisol trajectories between diagnostic groups underscore the relevance of salivary cortisol as a nuanced biomarker of stress regulation and physiological plasticity in pediatric populations. Future studies should consider complementing salivary cortisol data with additional stress markers and immune parameters to better delineate the biopsychosocial mechanisms involved. The results of the Six-Minute Walk Test are shown in Fig.\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eFigure 3. Summary results of the Six-Minute Walk Test (6MWT)\u003c/p\u003e\u003cp\u003eDgmain: F\u0026thinsp;=\u0026thinsp;83.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.738; Stage: F\u0026thinsp;=\u0026thinsp;11.7, p\u0026thinsp;=\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.116; GDM \u0026times; Stage: F\u0026thinsp;=\u0026thinsp;1.2, p\u0026thinsp;=\u0026thinsp;0.312, ηp2\u0026thinsp;=\u0026thinsp;0.0391; Subj(GDM): F\u0026thinsp;=\u0026thinsp;3.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.782; DE\u0026thinsp;\u0026lt;\u0026thinsp;BD, OB\u0026thinsp;\u0026lt;\u0026thinsp;BD, OB\u0026thinsp;\u0026lt;\u0026thinsp;RD, OB\u0026thinsp;\u0026lt;\u0026thinsp;DE,\u003c/p\u003e\u003cp\u003eLegend: BD \u0026ndash; nephrological indication group; RD \u0026ndash; respiratory disease indication group; DE \u0026ndash; dermatological indication group; OB \u0026ndash; obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e\u003cp\u003eFigure 4. Summary results of individual participant groups in the Four Square Step Test (FSST)\u003c/p\u003e\u003cp\u003eDgmain: F\u0026thinsp;=\u0026thinsp;1.1, p\u0026thinsp;=\u0026thinsp;0.384, ηp2\u0026thinsp;=\u0026thinsp;0.0341; Stage: F\u0026thinsp;=\u0026thinsp;36.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.236; GDM \u0026times; Stage: F\u0026thinsp;=\u0026thinsp;3.2, p\u0026thinsp;=\u0026thinsp;0.015, ηp2\u0026thinsp;=\u0026thinsp;0.098; ; Subj(GDM): F\u0026thinsp;=\u0026thinsp;6.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp2\u0026thinsp;=\u0026thinsp;0.867; RD2\u0026thinsp;\u0026lt;\u0026thinsp;RD1, OB2\u0026thinsp;\u0026lt;\u0026thinsp;OB1\u003c/p\u003e\u003cp\u003eLegend: HC \u0026ndash; control group; BD \u0026ndash; nephrological indication group; RD \u0026ndash; respiratory disease indication group; DE \u0026ndash; dermatological indication group; OB \u0026ndash; obesity indication group. Stage denotes testing at baseline (1) and post-treatment (2).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis pilot prospective study demonstrated that a 28-day spa treatment leads to measurable changes in salivary cortisol levels in children aged 9\u0026ndash;12 years with obesity and respiratory diseases. In obese children, a statistically significant decrease in cortisol was observed, which may be interpreted as a favorable hormonal response to weight reduction, likely associated with decreased production of proinflammatory mediators by adipose tissue. Weight loss appears to contribute to improved emotional well-being and to a broader enhancement of limbic system function, with downstream effects on key regulatory axes (psyche, immunity, metabolism).\u003c/p\u003e\u003cp\u003eIn contrast, an increase in cortisol levels was observed in children with chronic respiratory diseases, possibly reflecting heightened HPA axis activation triggered by structured physical activity and routine interventions that include adequate sleep.\u003c/p\u003e\u003cp\u003eAgility test results (FSST) showed a statistically significant improvement in children with respiratory diseases and those who were overweight or obese (Fig.\u0026nbsp;4). Comparable improvements were observed in the two remaining diagnostic groups; however, due to the small number of participants in these subgroups, the results could not be statistically confirmed. The Six-Minute Walk Test (6MWT) results demonstrated a stratification of physical fitness according to diagnostic group.\u003c/p\u003e\u003cp\u003eLimitations\u003c/p\u003e\u003cp\u003eGiven the pilot nature of this study and the limited size of some subgroups, the findings should be interpreted with caution. Nevertheless, the study paves the way for further research into the relationships between body composition, hormonal regulation, and therapeutic response within the context of pediatric spa treatment.\u003c/p\u003e\u003cp\u003eClinical Implications\u003c/p\u003e\u003cp\u003eSalivary cortisol can be used as a sensitive, non-invasive, and repeatable marker of neuroendocrine activity in children undergoing inpatient treatment.\u003c/p\u003e\u003cp\u003eIn children with obesity, a decrease in cortisol may indicate not only a reduction in stress burden but also a favorable metabolic response to changes in physical activity and lifestyle.\u003c/p\u003e\u003cp\u003eAn increase in cortisol among children with respiratory diseases may reflect reactivation of the HPA axis following prior pharmacotherapy or increased physical exertion and should be interpreted in the context of the individual clinical condition.\u003c/p\u003e\u003cp\u003eThese findings support the use of spa treatment as a comprehensive therapeutic environment that positively influences the health status of children with chronic illnesses.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBMI \u0026ndash; Body Mass Index\u003c/p\u003e\u003cp\u003eCSRRC \u0026ndash; Comprehensive Spa Rehabilitative Care\u003c/p\u003e\u003cp\u003eDE \u0026ndash; Dermatological Indication Group\u003c/p\u003e\u003cp\u003eFFM \u0026ndash; Fat-Free Mass\u003c/p\u003e\u003cp\u003eFSST \u0026ndash; Four Square Step Test\u003c/p\u003e\u003cp\u003eHPA axis \u0026ndash; Hypothalamic\u0026ndash;Pituitary\u0026ndash;Adrenal Axis\u003c/p\u003e\u003cp\u003eIN/OUT \u0026ndash; Initial and Final Measurement Points\u003c/p\u003e\u003cp\u003eOB \u0026ndash; Obesity Indication Group\u003c/p\u003e\u003cp\u003eOPLS/O2PLS \u0026ndash; Orthogonal Projections to Latent Structures / Bidirectional O2PLS\u003c/p\u003e\u003cp\u003eRD \u0026ndash; Respiratory Disease Indication Group\u003c/p\u003e\u003cp\u003eSpO₂ \u0026ndash; Peripheral Oxygen Saturation\u003c/p\u003e\n\u003cp\u003e6MWT – Six-Minute Walk Test\u003c/p\u003e\n\u003cp\u003ePNIE axis \u0026ndash; Psycho\u0026ndash;Neuro\u0026ndash;Immune\u0026ndash;Endocrine Axis\u003c/p\u003e\u003cp\u003eVC/FEV₁ \u0026ndash; Vital Capacity / Forced Expiratory Volume in 1 Second\u003c/p\u003e\u003cp\u003eHC \u0026ndash; Healthy Controls (Control Group)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e The study was performed in line with the principles of the Declaration of Helsinki. Approval was obtained from the Ethics Committee of the Institute of Endocrinology (Prague, Czech Republic). Written informed consent was obtained from all parents or legal guardians of the participating children prior to enrollment.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by MH CZ \u0026ndash; DRO (Institute of Endocrinology \u0026ndash; E\u0026Uacute;, 00023761).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.T. conceived the study, coordinated data collection, and drafted the main manuscript text.D.J. contributed to clinical data acquisition and patient management.M.H. performed statistical analyses and contributed to data interpretation.M.B. and L.M. conducted hormonal analyses of salivary cortisol and supervised laboratory procedures.L.M. also assisted with literature review, data organization, and manuscript editing.All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKautz MM (2021) Applications of psychoneuroimmunology models of toxic stress in prevention and intervention efforts across early development. Brain Behav Immun Health 16:100322\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVečeřov\u0026aacute;-Proch\u0026aacute;zkov\u0026aacute; A, Psychoneuroimunologie (2004) Acta Psychiatrica Postgradualia Bohemica 6(4):164\u0026ndash;175\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVečeřov\u0026aacute;-Proch\u0026aacute;zkov\u0026aacute; A (2006) Stres, eustres a distres. Intern\u0026iacute; Medic\u0026iacute;na pro Praxi 8(4):171\u0026ndash;174\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eV\u0026eacute;le F (2012) Vyšetřen\u0026iacute; hybn\u0026yacute;ch funkc\u0026iacute; z pohledu neurofyziologie: Př\u0026iacute;ručka pro terapeuty pracuj\u0026iacute;c\u0026iacute; v neurorehabilitaci. Praha: Triton; 222 s. ISBN: 978-80-7387-608-1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraham BL, Steenbruggen I, Miller MR, Barjaktarevic IZ, Cooper BG, Hall GL et al (2019) Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European Respiratory Society Technical Statement. Am J Respir Crit Care Med 200(8):e70\u0026ndash;e88\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePushparaj PN, Tserentsoodol N, Hammock BD (2008) Saliva collection for steroid analysis. Clin Biochem 41(10\u0026ndash;11):874\u0026ndash;887\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories (2002) ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 166(1):111\u0026ndash;117\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDite W, Temple VA (2002) A clinical test of stepping and change of direction to identify multiple falling older adults. Arch Phys Med Rehabil 83(11):1566\u0026ndash;1571\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeloun M, Hill M, Militk\u0026yacute; J, Kupka K (2000) Transformation in the PC-aided biochemical data analysis. Clin Chem Lab Med 38(6):553\u0026ndash;559\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeloun M, Hill M, Militk\u0026yacute; J, Vrb\u0026iacute;kov\u0026aacute; J, Stanick\u0026aacute; S, Škrha J (2004) New methodology of influential point detection in regression model building for the prediction of metabolic clearance rate of glucose. Clin Chem Lab Med 42(3):311\u0026ndash;322\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeloun M, Militk\u0026yacute; J, Hill M, Brereton RG (2002) Crucial problems in regression modelling and their solutions. Analyst 127(4):433\u0026ndash;450\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrygg J, Wold S (2002) Orthogonal projections to latent structures (O-PLS). J Chemom 16(3):119\u0026ndash;128\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSafarzadeh E, Shafiee-Kandjani AR, Ghalehgolab-Behbahan A, Ghaffari S, Vahedi H (2005) Determination of salivary cortisol in healthy children and adolescents. Acta Med Iran 43(4):295\u0026ndash;300\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"saliva, cortisol, child, balneology, biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-7481334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7481334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to examine the impact of a 28-day structured spa treatment on salivary cortisol levels in children aged 9\u0026ndash;12 years diagnosed with obesity or chronic respiratory diseases. The study included 95 children at spas and 38 children from a primary school (control group). Laboratory analyses were performed at the Institute of Endocrinology in Prague. In children with obesity (n\u0026thinsp;=\u0026thinsp;34), a decrease in mean cortisol levels from 3.32 to 2.36 nmol/l was observed. In children with respiratory diseases not receiving corticosteroid therapy (n\u0026thinsp;=\u0026thinsp;18), cortisol levels increased from 2.68 to 3.48 nmol/l. The control group showed an increase from 3.52 to 4.02 nmol/l (n\u0026thinsp;=\u0026thinsp;38). These findings highlight the relevance of salivary cortisol as a dynamic stress biomarker for pediatric rehabilitation research. The findings support the utility of salivary cortisol as a biomarker of physiological response to therapeutic and rehabilitative interventions in pediatric populations.\u003c/p\u003e","manuscriptTitle":"Change in Salivary Cortisol Levels in Children (Aged 9–12 Years) with Obesity and Respiratory Diseases during a 28-Day Spa Treatment: A Pilot Prospective Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 06:30:00","doi":"10.21203/rs.3.rs-7481334/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-16T07:12:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T05:55:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126481506859128040217497207701854860295","date":"2025-10-01T18:04:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T08:56:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36988559834055392897470062250625723866","date":"2025-09-21T17:05:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-09T19:13:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T07:13:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T07:00:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Pediatrics","date":"2025-08-28T14:34:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1e0018c5-4a55-441f-af90-070201762c2f","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:49:04+00:00","versionOfRecord":{"articleIdentity":"rs-7481334","link":"https://doi.org/10.1007/s00431-025-06723-3","journal":{"identity":"european-journal-of-pediatrics","isVorOnly":false,"title":"European Journal of Pediatrics"},"publishedOn":"2026-01-16 16:30:22","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-09-22 06:30:00","video":"","vorDoi":"10.1007/s00431-025-06723-3","vorDoiUrl":"https://doi.org/10.1007/s00431-025-06723-3","workflowStages":[]},"version":"v1","identity":"rs-7481334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7481334","identity":"rs-7481334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}