Hear rate (HR) is continuously recorded throughout the procedures with a Polar RS800CX portable device using a transmitter consisting of a stable polyamide case with electrodes attached to an elastic belt fixated to the chest of participants. Chronotropic control of the heart is achieved via the complex interplay of the sympathetic (SNS) and the parasympathetic (PNS) branches of the ANS. HR is under tonic inhibitory control (PNS dominance over SNS influences) [
50], and the PNS modulation of the HR is fast (timescale of milliseconds) and short-lived, while SNS effects are slow on the timescale of seconds [
51]. The recording and analysis of the sequence of time intervals between adjacent heartbeats – the inter-beat-interval (IBI in milliseconds) - is therefore the basis for the calculation of all the measures of heart rate variability (HRV). HRV is a reliable and readily available measure of parasympathetic nervous system activity that typically decreases after painful stimulation [
52]. The Polar RS800CX is capable to produce IBIs at sampling frequency of 1000 Hz, providing a temporal resolution of 1 ms for each R–R interval. Device-specific software (Polar ProTrainer 5) is used to transfer recordings to a PC. IBI data (.txt files) is exported and HRV is analyzed using Kubios HRV (Biosignal Analysis and Medical Imaging Group, University Kuopio, Finland, Version 2.0) [
53]. Different indices of HRV are subsequently derived for further analysis. Given our particular interest in vagal activity, the square root of the mean squared difference of successive NN intervals (RMSSD,
ms), the proportion of pairs of successive NNs that differ by more than 50 ms (pNN50, %), and the spectral power expressed as normalized units of the high-frequency (HFn.u.; 0.15–0.4 Hz) band derived using a autoregressive algorithm are obtained. In case of screwed distributions, HRV data will be log-transformed for further statistical analysis [
54]. We derive HR and HRV for different time segments in the course of the procedure. There is a 5 min baseline recording before hand-immersion in the CPT and a 10 min post-line recording after hand-removal from the CPT. In addition to the continuous assessment of HR and HRV, blood pressure (BP) is measured for a total of four times, initiated at least 5 min before each CPT and immediately after hand removal using an automated digital device (Braun ExactFit™ 3 BP6000). We use an estimation of systolic and diastolic BP values, in addition to mean arterial pressure (MAP), for further analysis.
We assess HPA axis response using salivary samples. Saliva cortisol is a reliable and valid proxy of free plasma cortisol levels [
55,
56], sensitive to changes in HPA axis activation due to stressors [
55] such as experimentally induced pain [
57], and has previously been utilised in numerous studies with children and adolescents [
58‐
60]. We collect four salivary samples; one prior to each CPT and one following 15 min after hand removal. Collection of saliva samples is carried out by having participants chew on a cotton role (Salivettes; Sarstedt, Numbrecht, Germany) for 2 min. After the collection of salvia, samples (at least 1000 μl salvia) are labelled with the participant ID and time-point of assessment, and then are directly deep-freezed at −20 ° C and stored until assay. Cortisol will be analysed at the Institute of Pharmacology of the Medical Faculty Heidelberg (Steroid Lab). For preparation, samples will be centrifugated for 7 min at 3000 rpm and 100 μl salvia per participant will be used for assay. The assay for the determination of salvia cortisol and serum cortisol is a special in-house assay with extraction and subsequent radioimmunoassay (RIA) developed by the Steroid Lab at the Institute of Pharmacology of the Medical Faculty Heidelberg. The range of the standard curve is from 2,5 to 5000 pg including a limit of detection of 5 pg/probe. The intra-assay coefficient of variation is below 10 % and inter-assay coefficient of variation is below 15 %.