Salivary Cortisol as a Surrogate Marker of Stress Response in Children Undergoing Laparoscopic Surgery with Low-pressure versus Standard-pressure Pneumoperitoneum

ABSTRACT

Purpose:

Salivary cortisol is a validated, non-invasive biomarker of hypothalamic–pituitary–adrenal axis activity and surgical stress. Evidence on perioperative cortisol kinetics in children undergoing laparoscopic surgery is limited. This study evaluated temporal salivary cortisol trends and compared stress responses between low- and standard-pressure pneumoperitoneum (PP) in children undergoing laparoscopic surgery.

Methods:

In this prospective randomized trial, 101 children scheduled for laparoscopic surgery were allocated to low-pressure PP (6-8 mmHg) or standard-pressure PP (9-11 mmHg) using block randomization. Salivary cortisol was measured preoperatively and at 6, 12, 24, 48, and 72 h postoperatively using enzyme-linked immunosorbent assay. Hemodynamic and respiratory parameters, pain scores, time to resume feeding, and hospital stay were recorded and analyzed.

Results:

Ninety-three children completed the study (low-pressure: n = 47; standard-pressure: n = 46). Baseline cortisol levels were comparable (median: 5.94 vs. 4.96 nmol/L, P = 0.662). Across the cohort, cortisol showed a biphasic pattern: A rise peaking at 12 h, decline at 24 h, and secondary increase at 48 h, remaining above baseline at 72 h. No significant differences were observed between groups, though the low-pressure group had slightly higher cortisol at 12 h, and the standard-pressure group at 48 h. Hemodynamic and ventilatory parameters remained stable, and recovery outcomes – including pain, feed resumption, and hospital stay – were similar.

Conclusion:

Salivary cortisol is a reliable, noninvasive marker of perioperative stress in children undergoing laparoscopic surgery with a consistent postoperative trajectory. Within 6–11 mmHg, PP pressure did not influence stress response, intraoperative physiology, or short-term recovery.

INTRODUCTION

Pneumoperitoneum (PP) is an essential component of laparoscopic surgery that facilitates surgical access. However, insufflation of carbon dioxide (CO₂) causes peritoneal stretching and systemic absorption, which may alter homeostatic, metabolic, and cardiorespiratory parameters.[1,2,3,4] In pediatric patients, intra-abdominal pressure is lower than in adults, typically ranging from 5 to 7 mmHg. Nevertheless, laparoscopic procedures in children often employ PP pressures up to 12 mmHg, depending on age, weight, and body size.

A randomized study from our institution by Joon et al. demonstrated that higher PP pressures in young children were associated with increased CO₂ retention and greater postoperative analgesic requirements, without offering any additional benefit to the surgeon in terms of working space.[5] While these findings support the safety of performing pediatric laparoscopy at low-pressure PP, the broader effects of PP pressure on the perioperative stress response remain unclear, highlighting the need to investigate whether low-pressure PP attenuates stress in children undergoing laparoscopy.

Cortisol is the principal biomarker of physiological stress, measurable in serum, urine, sweat, and saliva.[6,7,8,9,10] Salivary cortisol reflects the unbound, biologically active fraction of circulating cortisol and has emerged as a noninvasive and reliable indicator of acute stress, particularly in the pediatric population.[10,11] Compared with serum cortisol, salivary cortisol offers several practical advantages – it is less invasive to collect, more acceptable to children and parents, and highly responsive to perioperative changes – making it an ideal marker for assessing surgical stress in children undergoing laparoscopy.[12]

Given the limited literature evaluating perioperative stress in pediatric laparoscopic surgery and the uncertain impact of PP pressure variation, this study aims to assess the perioperative stress response in children undergoing laparoscopic surgery using salivary cortisol as a surrogate biomarker. It also seeks to determine whether low-pressure PP provides a measurable advantage over standard-pressure PP in attenuating the surgical stress response in children.

METHODS

Study design and ethical approval

This prospective, randomized clinical trial was conducted at a tertiary care teaching institute in North India between November 2023 and May 2025. The study was approved and monitored by the institutional ethics committee (IEC Code: 2023/299/MCh-135) and conducted in accordance with the principles of the 1964 Helsinki Declaration and its subsequent amendments. Written informed consent was obtained from parents or legal guardians before enrollment. The study was supported by an intramural research grant (PGI/DIR/RC/01/2024) and registered with the Clinical Trials Registry-India (CTRI/2024/10/075869).

This randomized controlled trial was conducted and reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) guidelines for randomized trials. A CONSORT flow diagram detailing participant screening, randomization, follow-up, and final analysis is presented in [Figure 1].

Figure 1.

Study methodology (Hrs: hours, HR: Heart rate, MAP: Mean arterial pressure, EtCO2: End tidal CO2, T0: before insufflation, T1: 20 min after insufflation, T2: before desufflation, T3: 10 min after desufflation)

Participants

Patients aged 1–18 years undergoing laparoscopic surgery at our center were eligible for inclusion [Figure 1]. Exclusion criteria were underlying adrenal pathology (e.g., adrenal tumors, congenital adrenal hyperplasia), ongoing steroid therapy for any indication, and laparoscopic procedures for acute inflammatory conditions such as appendicitis or Meckel’s diverticulitis.

Randomization

Participants were allocated into two groups using a block randomization method with variable block sizes of 2 and 4 to ensure balanced distribution throughout enrolment. A total of 101 patients were randomized according to a pre-generated allocation sequence. Each patient’s sequence number determined group assignment, and the corresponding PP pressure was applied in accordance with the allocated group [Figure 1].

Anesthesia protocol

Upon transfer to the operating theatre, standard monitoring was applied, including a five-lead electrocardiogram, automated non-invasive blood pressure, and pulse oximetry. Anesthesia was induced with thiopental sodium 4 mg/kg and fentanyl citrate 3 μg/kg. Following adequate preoxygenation, tracheal intubation was facilitated with atracurium besylate 0.5 mg/kg. Anesthesia was maintained with sevoflurane in an air/O₂ mixture (FiO₂ = 0.4) using volume-controlled ventilation at an I:E ratio of 1:2. Children were ventilated with a tidal volume of 8 mL/kg and a positive end-expiratory pressure of 3 cmH₂O.

Surgical technique

After induction of anesthesia, the first port was inserted under direct vision using the open technique. The abdomen was insufflated with CO₂ using an electronic insufflator (Karl Storz GmbH and Co. KG, Tuttlingen, Germany) at an initial flow rate of 0.5–1 L/min to achieve the target pressure according to group allocation (low-pressure PP: 6–8 mmHg or standard-pressure PP: 9–11 mmHg). The laparoscopic procedure was subsequently performed.

Hemodynamic monitoring and perioperative data

Hemodynamic and respiratory parameters, including heart rate (HR), noninvasive mean arterial pressure (MAP), and end-tidal CO₂ (EtCO₂), were recorded at four time points: before CO₂ insufflation (T0), 20 min after insufflation (T1), before desufflation (T2), and 10 min after desufflation (T3). At the end of the procedure, neuromuscular blockade was reversed with intravenous neostigmine 50 μg/kg and glycopyrrolate 30 μg/kg. The duration of surgery, length of hospital stay, time to resumption of oral feeding, and postoperative pain scores were documented. Pain was assessed at 24 and 48 h postoperatively using a Visual Analog Scale (VAS) ranging from 0 to 10.[13]

Saliva collection and preparation

Saliva samples (0.5–1 mL) were collected from each participant preoperatively and at 6, 12, 24, 48, and 72 h postoperatively. The 6-h postoperative sample was collected on the day of surgery, typically between 21:00 and 24:00 h, whereas the 12-h postoperative sample was collected the following morning between 06:00 and 09:00 h. Children were instructed to pool saliva in the mouth and drool it passively into the receiving container. Unstimulated saliva samples were refrigerated at 4°C within 30 min and frozen at or below –20°C within 12 h of collection.

Precautions before collection included avoiding major meals for at least 1 h, refraining from sugary drinks within 20 min, rinsing the mouth with water but not within 10 min, and avoiding tooth brushing before collection.

Cortisol assay

Salivary cortisol concentration was measured in each sample using a Salivary Cortisol enzyme-linked immunosorbent assay kit (SLV-2930), following the manufacturer’s instructions. The assays were performed by laboratory personnel who were blinded to the study group allocation.

Sample size

Based on the study by Grigoropoulou et al.,[14] the mean reduction in salivary cortisol levels was 59.81% in the unexplained group (14.78–5.94) and 87.97% in the explained group (13.38–1.61). Using these differences (59.81% vs. 87.97%), with a two-sided 95% confidence interval and 80% power, the required sample size was 37 participants per group. To account for potential dropouts, 80 consecutive eligible participants were planned for enrolment and randomized equally between the two groups (40 patients per group). Sample size estimation was performed using G*Power software version 3.1.9.7. (G*Power 3.1.9.7 (Heinrich-Heine-Universität, Düsseldorf, Germany)).

Statistical analysis

Continuous variables were presented as median with interquartile range (IQR), as the data were non normally distributed, evaluated using Kolmogorov–Smirnov test. Mann–Whitney U test was used to compare the medians between the two groups. Categorical variables were presented in numbers (%) and compared by the Chi-square test. The Friedman test for one-way ANOVA was used to compare the medians in the same groups over time. To test the association between the study groups and the change in the median values over time, the Friedman test for two-way ANOVA was used. As data had some missing values during the follow-up, alternatively, linear mixed-effects modeling was performed to evaluate changes in stress response across six time points between treatment groups for changing the effect between treatments, time points, and interactions between them. Spearman’s rank correlation coefficient was used to assess the linear relationship between salivary cortisol and demographic and hemodynamic variables observed over time. All statistical analyses were performed using SPSS software for Windows. version 23.0 (SPSS, Chicago, IL, USA) and STATA-16. P <0.05 was considered statistically significant.

RESULTS

A total of 101 children were enrolled in the study, of whom 8 were excluded due to insufficient salivary samples, leaving 93 participants for the final analysis. Among these, 47 underwent laparoscopic surgery with low-pressure PP (6–8 mmHg) and 46 with standard-pressure PP (9–11 mmHg) [Figure 1].

Demographic characteristics, including age, sex, weight, height, and primary diagnosis, were comparable between the two groups (all P > 0.05). The median age of the cohort was 112 months (IQR: 63–153), and 62.4% were male. The low-pressure PP group comprised 30 males and 17 females, while the standard-pressure PP group included 28 males and 18 females (P = 0.927). The median weight was 25 kg (IQR: 17–42), and the median height was 127 cm (IQR: 110–152). Surgical indications were gastrointestinal pathologies in 16 patients (17.2%), hepatobiliary disorders in 40 (43%), and urological conditions in 37 (39.8%) [Tables 1 and 2].

Table 1.

Comparison of variables between low and standard pressure pneumoperitoneum groups (n=93)

Variables	Entire cohort (n=93)	Low-pressure PP (n=47)	Standard-pressure PP (n=46)	P
Demographic data
Age (months)	112 (63–153)	115 (76-153)	110 (61-158)	0.927
Male	58 (62.4)	30 (63.83)	28 (60.87)	0.768
Female	35 (37.6)	17 (36.17)	18 (39.13)
Weight (kg)	25 (17–42)	27 (16–44)	23 (17–42)	0.857
Height (cm)	127 (110–152)	127 (105–150)	125 (110–154)	0.727
Organ system distribution of primary diagnosis
Gastrointestinal	16 (17.2)	8 (17.02)	8 (17.39)	0.847
Hepato-biliary	40 (43)	19 (40.43)	21 (45.65)
Urology	37 (39.8)	20 (42.55)	17 (36.96)
Intra-operative parameters
Duration of surgery (min)	150 (109–232)	137 (103–200)	165 (110–245)	0.518
HR (beats per min)
HR T0	90 (81–110)	90 (76–106)	91.5 (82–110)	0.456
HR T1	100 (84–110)	100 (82–110)	104.5 (84–110)	0.684
HR T2	100 (84–114)	97 (84–110)	107 (85–118)	0.523
HR T3	100 (88–112)	100 (82–117)	102.5 (90–110)	0.617
MAP (mmHg)
MAP T0	70 (62–80)	70 (62–80)	70 (62–78)	0.669
MAP T1	74 (67–80)	73 (67–80)	74.5 (66–81)	0.631
MAP T2	73 (67–78)	70 (67–78)	74 (68–79)	0.435
MAP T3	73 (68–81)	72 (68–80)	75 (68–82)	0.508
EtCO2 (mmHg)
EtCO2 T0	34 (33–36)	34 (33–36)	35 (33–36)	0.831
EtCO2 T1	37 (36–39)	37 (35–40)	37 (36–39)	0.898
EtCO2 T2	37 (35–38)	37 (35–38)	37 (35–38)	0.997
EtCO2 T3	35 (33–36)	35 (33–37)	35 (34–36)	0.469
Salivary cortisol levels (nmol/L)
Preoperative	5.32 (2.33–11.07)	5.94 (1.77–12.81)	4.96 (2.8–10.13)	0.662
6 h postoperative	7.41 (4.67–19.33)	6.85 (4.3–24.99)	7.79 (5.3–16.16)	0.811
12 h postoperative	8.43 (3.63–18.7)	12.05 (3.23–19.48)	6.47 (4.25–14.71)	0.218
24 h postoperative	5.06 (3.15–11.92)	6.17 (2.22–12.66)	4.96 (3.44–10.13)	0.857
48 h postoperative	8.74 (4.05–16.02)	9.11 (4.05–20.34)	7.93 (4.04–13.66)	0.796
72 h postoperative	8.7 (4.08–13.17)	8.82 (6.3–15.63)	7.38 (3.15–12.85)	0.179
Postoperatively parameters
Time to resume feeding (days)	1 (1–2)	1 (1–2)	1 (1–1)	0.181
Postoperative pain 24 h	4 (3–4)	4 (3–4)	4(3–4)	0.286
Postoperative pain 48 h	2 (1–3)	2 (1–3)	2 (1–3)	0.687
Length of hospital stay (days)	4 (4–6)	5 (4–7)	4(4 -5)	0.108

Data is presented as median (IQR) and compared using the Mann–Whitney U-test, or as n (%) and compared using the Chi-square test. P<0.05 is considered significant. Postoperatively, the pain score was assessed according to VAS. PP: Pneumoperitoneum, T0: Before insufflation, T1: 20 min after insufflation, T2: Before desufflation, T3: 10 minutes after desufflation, IQR: Interquartile range, VAS: Visual Analog Scale, MAP: Mean arterial pressure, HR: Heart rate, EtCO2: End tidal CO₂

Table 2.

Distribution of primary diagnosis between study groups

Diagnosis	Low-pressure PP	Standard-pressure PP
Gastrointestinal	n=8	n=8
Achalasia cardia	1	1
PFIC	0	1
Chronic appendicitis	3	1
Malrotation	1	1
Hirschsprung’s disease	2	2
Mesenteric cyst	1	2
Hepato-biliary	n=19	n=21
Cholelithiasis	16	19
Choledochal malformation	3	2
Urology	n=20	n=17
Hernia/UDT/DSD	13	10
PUJO/Duplex Kidney	6	5
MCDK	1	2

PP: Pneumoperitoneum, PFIC: Progressive familial intrahepatic cholestasis, UDT: Undescended testis, DSD: Disorders of sex development, PUJO: Pelvic ureteric junction obstruction, MCDK: Multicystic dysplastic kidney

The median duration of surgery for the entire cohort was 150 min (IQR: 109–232), with no significant difference between the low-pressure and standard-pressure PP groups (137 vs. 165 min; P = 0.518). Intraoperative monitoring of hemodynamic and respiratory parameters showed that HR, MAP, and EtCO₂ remained stable across all time points in both groups. At baseline (T0), median HR, MAP, and EtCO₂ were comparable, followed by a mild increase after insufflation (T1). Values remained stable during PP (T2) and returned toward baseline after desufflation (T3). Across all four time points, there were no statistically significant differences between the groups (all P > 0.05) [Table 1 and Figure 2a-c].

Figure 2.

Temporal trends of (a) heart rate, (b) mean arterial pressure, and (c) end-tidal CO₂ between low-and standard-pressure pneumoperitoneum groups (PP: Pneumoperitoneum, T0: Before insufflation, T1: 20 min after insufflation, T2: Before desufflation, T3: 10 min after desufflation)

Salivary cortisol levels in the entire cohort increased after laparoscopic surgery, reflecting a postoperative stress response. Levels rose from baseline to 6 h, peaked at 12 h, and then gradually declined by 24 h. A secondary rise was observed at 48 h, followed by a slight decrease at 72 h; however, values remained above preoperative levels. While the overall trend was consistent across groups, the patterns showed minor differences between the low-pressure and standard-pressure PP groups [Table 1 and Figure 3a and b].

Figure 3.

Temporal trends of salivary cortisol levels (nmol/L): (a) Entire cohort and (b) Comparison between low-and standard-pressure pneumoperitoneum groups (PP: Pneumoperitoneum, Hrs: Hours, Pre-op: Preoperatively, Post-op: Postoperatively)

Preoperative salivary cortisol concentrations, measured on the day before surgery, were comparable between the two groups. The median baseline salivary cortisol level was 5.94 nmol/L (IQR: 1.77–12.81) in the low-pressure PP group and 4.96 nmol/L (IQR: 2.80–10.13) in the standard-pressure PP group, with no statistically significant difference (P = 0.662) [Table 1 and Figure 3b].

Comparison of the stress response between the two groups showed no statistically significant differences at any postoperative time point, although median salivary cortisol levels were consistently higher in the low-pressure PP group than in the standard-pressure PP group. At 12 h, the peak postoperative level was 12.05 nmol/L in the low-pressure PP group compared with 6.47 nmol/L in the standard-pressure PP group (P = 0.218). At 48 h, median salivary cortisol was slightly higher in the standard-pressure group, suggesting a tendency toward a more prolonged stress response. Salivary cortisol levels did not return to baseline even at 72 h postoperatively, reflecting the persistence of a postoperative stress response [Table 1 and Figure 3b].

Friedman one-way repeated-measures analysis confirmed a temporal trend within the entire cohort and within each group (P = 0.223 for the entire cohort, P = 0.607 for the low-pressure PP group, and P = 0.326 for the standard-pressure PP group) [Table 3]. Friedman’s two-way repeated-measures analysis of variance revealed no significant interaction between group and time (P = 0.064), indicating that the overall pattern of stress response was similar regardless of PP pressure. As data have some missing values during the follow-up, alternatively, linear mixed-effects modeling was performed to evaluate changes in stress response across six time points between treatment groups. The analysis showed no significant effect of time on stress response (P = 0.784). There was also no significant difference between treatment groups (P = 0.499). In addition, the time × treatment interaction was not statistically significant, indicating that the trajectory of stress response over time was similar between the two groups (P = 0.664) [Table 3].

Table 3.

Comparison of salivary cortisol levels (nmol/L) at different time points between pneumoperitoneum groups (n=93)

PP Groups	Salivary cortisol levels (nmol/L) at different time points						P	P*
	Preoperatively	6 h postoperatively	12 h postoperatively	24 h postoperatively	48 h postoperatively	72 h postoperatively
Entire cohort (n=93)	5.32 (2.33–11.07)	7.41 (4.67–19.33)	8.43 (3.63–18.7)	5.06 (3.15–11.92)	8.74 (4.05–16.02)	8.7 (4.08–13.17)	0.223	-
Low-pressure PP (n=47)	5.94 (1.77–12.81)	6.85 (4.3–24.99)	12.05 (3.23–19.48)	6.17 (2.22–12.66)	9.11 (4.05–20.34)	8.82 (6.3–15.63)	0.607	0.064
Standard-pressure PP (n=46)	4.96 (2.8–10.13)	7.79 (5.3–16.16)	6.47 (4.25–14.71)	4.96 (3.44–10.13)	7.93 (4.04–13.66)	7.38 (3.15–12.85)	0.326

Data is presented as median (IQR), To compare the change in median scores over the time in the same group - one way Friedman repeated-measure ANOVA was used. To compare the change in median scores over time between the two groups - Friedman two way repeated-measure ANOVA was used. P<0.05 was considered significant. PP: Pneumoperitoneum, IQR: Interquartile range, ANOVA: Analysis of variance

No intraoperative or postoperative complications were observed in either group during the study period. There were no significant differences between the two groups in postoperative recovery outcomes. The median time to resumption of oral feeding was 1 day (IQR: 1–2) in the low-pressure PP group and 1 day (IQR: 1-1) in the standard-pressure PP group (P = 0.181). Postoperative pain scores were comparable at both 24 h (median 4 [IQR: 3–4] vs. 4 [IQR: 3–4]; P = 0.286) and 48 h (median 2 [IQR: 1–3] vs. 2 [IQR: 1–3]; P = 0.687). The median duration of hospital stay was 5 days (IQR, 4-7) in the low-pressure PP group and 4 days (IQR: 4–5) in the standard-pressure PP group (P = 0.108) [Table 1].

Linear correlation analysis was performed between salivary cortisol levels and age, weight, height, duration of surgery, pain scores at 24 and 48 h postoperatively, time to resume oral feeds, and length of hospital stay. The analysis showed only weak correlations (r < 0.4). Statistically significant associations were observed between height and salivary cortisol level at 6 h postoperatively (P = 0.04), and between length of hospital stay and the change in salivary cortisol from baseline to 12 h postoperatively (P = 0.031) [Table 4].

Table 4.

Linear correlation of variables with salivary cortisol levels (n=93)

Variables	Salivary cortisol level						Change in salivary cortisol levels between preoperative and 12 h postoperative
	Preoperative		6 h postoperative		12 h postoperative
	Correlation coefficient	P	Correlation coefficient	P	Correlation coefficient	P	Correlation coefficient	P
Age	0.085	0.44	0.192	0.14	0.03	0.81	0.016	0.896
Weight	−0.006	0.96	0.248	0.05	0.011	0.93	0.061	0.62
Height	0.042	0.7	0.263	0.04	0.034	0.78	0.041	0.739
Duration of surgery	-	-	−0.093	0.47	0.076	0.53	0.106	0.388
Time to resume feeding (days)	-	-	0.013	0.92	0.07	0.56	0.06	0.628
Postoperatively pain 24 h	-	-	−0.084	0.52	0.057	0.63	0.157	0.2
Postoperatively pain 48 h	-	-	−0.123	0.34	0.053	0.66	0.095	0.44
Length of hospital stay (days)	-	-	0.035	0.79	0.182	0.13	0.262	0.031

Data are presented as Spearman’s rho correlation coefficient. P<0.05 is considered significant

DISCUSSION

This study provides new insights into baseline and postoperative temporal trends of salivary cortisol in children undergoing laparoscopic surgery, using it as a noninvasive biomarker to characterize the neuroendocrine stress response under low-pressure PP (6–8 mmHg) and standard-pressure PP (9–11 mmHg). Our findings establish reference preoperative salivary cortisol values and reveal a consistent postoperative trajectory, marked by an early rise with peak levels at 12 h and only partial decline by 72 h. These observations add to the growing body of evidence on perioperative stress in children and may help develop strategies to optimize stress modulation in pediatric laparoscopic surgery.

In our study, intraoperative monitoring showed that HR, MAP, and EtCO₂ had insignificant variation across all measured time points in both low-pressure and standard-pressure PP groups. These findings align with those of Shoar et al., who, in a randomized double-blind adult trial, also reported no significant differences in HR or MAP between low-and standard-pressure PP during laparoscopic cholecystectomy, although a nonsignificant upward trend was noted in the standard-pressure group.[15]

Our findings partly contrast with those of Joon et al., who, in a randomized trial of children aged 1–5 years at our institution, reported significant rises in PaCO₂ and EtCO₂ in the standard-pressure group at T1 and T2, despite no major hemodynamic changes at 9–10 mmHg PP.[5] Similarly, Hou et al., in a retrospective series of 1776 children undergoing laparoscopic hernia repair, observed significant intraoperative increases in blood pressure and PaCO₂ after CO₂ insufflation.[16] In our cohort, however, EtCO₂, used as a non-invasive surrogate for PaCO₂, showed only minimal, nonsignificant changes, suggesting adequate ventilatory compensation.

This study defines baseline salivary cortisol levels and their temporal trends in children aged 1–18 years undergoing laparoscopic surgery. The cohort’s median baseline salivary cortisol level was 5.32 nmol/L (IQR: 2.33–11.07), consistent with the physiological reference range in the pediatric population, with no significant intergroup difference. Post-operatively, salivary cortisol levels followed a characteristic biphasic pattern: an initial post-operative rise peaking at 12 h, a decline by 24 h, and a secondary elevation at 48 h, with levels remaining above baseline even at 72 h. This temporal trend reflects an expected activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to surgical stress, with sustained elevation suggesting a prolonged physiological recovery period.

Our findings are consistent with Adachi et al., who reported a significant rise in salivary cortisol after elective pediatric surgery, returning to baseline by postoperative day 3, reinforcing its value as a noninvasive biomarker of surgical stress.[6] In contrast, Tasar et al. observed a blunted response in children undergoing congenital heart surgery, likely due to HPA axis dysregulation from underlying cardiac pathology, underscoring the influence of patient population and underlying disease on perioperative cortisol dynamics.[17] Similarly, Volkan et al. found no difference in preprocedural salivary cortisol levels in children undergoing gastroscopy, regardless of preparatory explanation.[18] This supports the robustness of salivary cortisol levels as a stable baseline marker in children and its sensitivity to procedural stress thereafter. Our study also confirms the utility of salivary cortisol levels as a reliable surrogate marker of stress response in otherwise healthy children undergoing laparoscopic surgery, and its capacity to reflect postoperative neuroendocrine stress response.

This study found no statistically significant differences in salivary cortisol between low- and standard-pressure PP groups at any postoperative time point. The low-pressure group showed slightly higher cortisol during the early post-operative period, peaking at 12 h, while the standard-pressure group demonstrated a modest secondary rise at 48 h, suggesting a tendency toward prolonged stress. However, these variations were not significant. Both groups followed a similar temporal pattern, with a peak at 12 h and persistent elevation above baseline through 72 h. Repeated-measures analysis confirmed no group-time interaction, indicating comparable physiological stress responses across PP pressures.

These findings align with Shoar et al., who reported no significant differences in systemic stress markers, including cortisol, between adult patients undergoing laparoscopic cholecystectomy at low versus standard PP pressures.[15] Similarly, Grigoropoulou et al. and Volkan et al. emphasized that perioperative salivary cortisol in children is influenced more by psychological preparedness and procedural context than by minor technical variations.[14,18] In contrast, Niu et al. observed significantly higher intraoperative serum cortisol in children undergoing surgery with high-pressure PP (12 mmHg) compared to low-pressure PP (8 mmHg), though levels normalized by 12 h.[19] Differences from our findings may relate to methodological factors, including salivary versus serum cortisol measurement, lower PP ranges in our study (9–11 mmHg vs. 12 mmHg), and variability in surgical complexity and duration.

Adachi et al. further highlighted the value of salivary cortisol as a stress marker, showing significantly lower levels after minimally invasive – particularly robotic-assisted – procedures compared with open surgery in 85 children undergoing elective operations. They also noted that prolonged operative time only transiently elevated cortisol, suggesting that while duration may briefly amplify stress, the surgical approach exerts a more sustained influence.[6] These results are consistent with our findings, reinforcing that less invasive techniques, even when differing in PP pressures and operative duration, tend to provoke comparable self-limiting stress responses.

Larsen et al. reported that gasless laparoscopy reduced intraoperative cortisol compared with conventional CO₂ PP, though post-operative inflammatory markers were paradoxically higher, suggesting that endocrine stress modulation may not directly improve recovery.[20] Karišik et al. further emphasized the role of non-technical factors – such as gender and psychosocial context – in shaping pediatric stress responses, which may partly account for the interindividual variability observed in our cohort.[21]

In this study, postoperative recovery outcomes – including time to oral feeding, pain scores, and hospital stay – were comparable between children undergoing laparoscopic surgery with low-versus standard-pressure PP. Median time to feeding was 1 day in both groups, and pain scores at 24 and 48 h showed no significant difference. Length of stay was also similar, indicating that within the studied pressure range (6–11 mmHg), PP pressure did not affect early recovery in pediatric patients.

Our results align partly with Perrakis et al., who reported no significant differences in post-operative pain between pressure groups.[22] However, Joon et al. observed consistently higher pain scores and analgesic requirements in children operated on under standard-pressure PP, particularly in the first 12 h.[5] Similarly, Vijayaraghavan et al. demonstrated lower pain scores across all time points in adults undergoing low-pressure PP, supporting the view that reduced insufflation pressure may attenuate peritoneal irritation and related pain.[23]

Further evidence of the link between stress physiology and post-operative discomfort comes from Adachi et al., who found that elevated salivary cortisol correlated with higher pain scores on postoperative days two and three.[6] This highlights salivary cortisol as both a biomarker of stress and an indirect indicator of nociception. Although our study did not identify significant intergroup pain differences, persistently elevated cortisol suggests ongoing stress activation, which may carry subclinical implications for recovery. Pediatric pain perception and expression are shaped by multiple intrinsic and extrinsic factors. Karišik et al. noted that gender, emotional regulation, and sociocultural influences – such as parental anxiety – can significantly modulate how children express pain, particularly in the early postoperative period.[21] This variability may obscure the isolated impact of PP pressure on pain outcomes, highlighting the complexity of interpreting recovery in pediatric populations.

This study makes a novel contribution by establishing baseline salivary cortisol levels in children and defining their postoperative temporal trends under varying PP pressures. While prior evidence, including systematic reviews of dexamethasone, highlights the effectiveness of pharmacologic interventions in attenuating inflammatory markers,[24] our results show that altering PP pressure alone does not significantly influence the neuroendocrine stress response. These findings suggest that pressure adjustment by itself is insufficient to modulate stress physiology, underscoring the need for multimodal strategies. Importantly, our study provides a reference framework for future pediatric trials evaluating both surgical and adjunctive approaches to optimize recovery.

The strengths of this study include its randomized controlled, prospective design with sample size determined through prior statistical calculation, as well as the use of standardized perioperative protocols, enhancing both reliability and generalizability. Serial salivary cortisol measurements over 72 h offered valuable insight into both acute and subacute neuroendocrine dynamics. The application of comprehensive statistical methods – including linear correlation and temporal trend analyses – allowed for a nuanced evaluation of the stress response and its association with perioperative factors.

Several limitations should be noted. The inclusion of a heterogeneous mix of surgical procedures with varying complexity and duration may have introduced physiological variability. Inadequate salivary volumes in some cases required participant exclusion, and logistical constraints prevented uniform sampling at identical time points, potentially contributing to variability. Moreover, additional inflammatory and metabolic biomarkers (e.g., interleukin-6, C-reactive protein, and catecholamines) were not assessed, which could have provided complementary perspectives on the stress response. Finally, long-term outcomes, including late recovery and potential neuroendocrine sequelae, were beyond the scope of this study.

CONCLUSION

This randomized study establishes salivary cortisol as a reliable, non-invasive marker of perioperative stress in children undergoing laparoscopic surgery. Cortisol levels followed a consistent post-operative trajectory, peaking at 12 h and remaining elevated above baseline levels at 72 h. Comparisons of low-and standard-pressure PP showed no significant differences in cortisol response, intra-operative hemodynamic and respiratory parameters, or recovery outcomes, indicating that pressures within 6–11 mmHg are safe, well tolerated, and have minimal impact on short-term recovery. These findings provide a reference framework for pediatric laparoscopy and support future research incorporating larger cohorts, longer follow-up, and additional biomarkers to better characterize the multidimensional stress response.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.