Secondary outcome
Salivary cortisol levels, the secondary outcome of this study, will be measured once at admission on day 1 at 21:00, and three times daily on days 2–4; for example, cortisol will be measured within 30 min of waking, at 12:00, and at 21:00. Subjects will not eat within approximately 1 h or brush their teeth within 10 min before saliva collection in order to avoid contamination.
Blood samples for analysis of epinephrine, norepinephrine, oxidative stress-related biomarkers (reactive oxygen species (ROS), nitric oxide (NO), malondialdehyde (MDA), protein carbonyl, glutathione (GSH), GSH reductase (GSH-Rx), superoxide dismutase (SOD), catalase, and total antioxidant capacity (TAC)), homocysteine, and immunological factors (tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-10, IL-12, T cells, B cells, and natural killer (NK) cells) will be collected at the same time as the serum cortisol samples. Complete blood counts will be conducted to quantify the populations of T cells, B cells, and NK cells during phase II, day 2.
A self-reported questionnaire validated for each purpose and modified to a Korean version will be utilised to measure perceived fatigue. The FSS will be used to measure overall fatigue status [
14], the BFI for variation in daily fatigue [
15,
16], and the LSEQ for variation in sleep aspects [
17]. The nine-item FSS is a self-reporting questionnaire developed by Krupp et al. [
14] and serves to evaluate fatigue during the past week using statements that the participants must scale from 1 to 7 in terms of degree of agreement. The BFI is a self-reporting questionnaire developed by Mendoza et al. [
15] to evaluate fatigue in patients with cancer. This nine-item questionnaire assesses the degree of fatigue at the present moment and for the past 24 h, as well as the impact of fatigue on daily life, on a scale of 0 to 10. The LSEQ consists of 10 items, each of which is measured on the Visual Analogue Scale (VAS); each item contains opposite statements at both extremes. The LSEQ involves four domains related to sleep: ease of initiating sleep (questions 1, 2, and 3), quality of sleep (questions 4 and 5), ease of waking (questions 6 and 7), and behaviour after waking (questions 8, 9, and 10). To complement the results of the BFI-K and the KMLSEQ, a daily sleep and fatigue status questionnaire developed by our research team will also be completed by the participants. This checks daily sleep and fatigue intuitively on the VAS and is structured so that participants can spontaneously describe a variety of their own symptoms.
In the afternoon of days 1–4, HRV testing will be performed to study variations in the autonomic nervous system responses relevant to fatigue, where HRV is measured as variations in the beat-to-beat intervals; R is a point corresponding to the peak of the QRS complex of the electrocardiogram (ECG) wave and RR is the interval between successive Rs. For HRV assessments, 5 min of ECG signalling will be measured and calculated using a Neo Dinamika system (MR Co. Ltd., Gyeonggi-do, Republic of Korea). The HRV measurements will be taken after participants rest for 10 min, and the subjects will be seated comfortably in a chair in a quiet and bright room at 20–25 °C for the assessment. The equipment will be connected to the medial side of both wrists and ankles as well as to the insides of both wrists and the right ankle. Rhythm parameters of the HRV include pulse rate, vegetative balance index, rhythm vegetative factor, regulation process adequacy index, and tension index. Vegetative regulation parameters of the HRV include regulation level and regulation reserves. Statistical analysis of the HRV includes the RRNN (mean of normal-to-normal (NN) intervals, which is all intervals between adjacent QRS complexes); the SDNN (standard deviation of NN intervals); CV (coefficient variation); the RMSSD (the root mean square of successive NN interval differences); the NN50 (the number of interval differences of successive NN intervals greater than 50 ms); and the pNN50 (the proportion of NN50 divided by total number of the mean of all normal NN intervals). Spectral analysis includes HF (high frequency, 0.15–0.4 Hz), LF (low frequency, 0.04–0.15 Hz), VLF (very low frequency, 0.0033 to 0.04 Hz), normalised HF, normalised LF, LF/HF ratio and TP (total power).