Selections of participants and study design

Five young analog astronauts (4 males and 1 female), whose characteristics are reported in Table 1, were recruited among the sixth Euro Moon Mars Poland analog astronaut mission (EMMPOL 6) in the Analog Astronaut Training Center (AATC Poland) as a part of the Euro Moon Mars projects by International Lunar Exploration Working Group (ILEWG).

Table 1 Anthropometric parameters collected from the subjects

The subjects that voluntarily took part in this mission, were asked to stay for one week inside an Earth-based moon-settlement-like habitat. The habitat was composed of two dining/working rooms, one bedroom, one bathroom and a small gym. The total surface of the habitat was about 57 square meters. The habitat had no sources of natural light or ventilation, therefore, for the whole duration of the mission the subjects were always exposed to artificial light, even during the sleep time, and poor air quality with increased levels of CO2. Each subject had a role inside the mission: commander, vice-commander, crew medical officer, data officer and communication officer and were asked to perform and complete a personal experiment during the week. Other tasks included permanent experiments that are in course in the habitat and are maintained by each crew and other 1-week experimental protocols whose topics ranged from geology, biology, botany, engineering, astronomy, and psychology. All the activities were strictly regulated in a schedule defined by the mission control group, which monitored the mission from the outside of the habitat. During the mission, subjects run on “mission time” that started from when they entered the habitat, losing contact with the normal time course. Sleep and wake time were decided by the mission control, and they were progressively reduced during the mission and shifted from normal nocturnal sleeping time creating a shift in normal circadian rhythms that are connected to light and dark cycles. The analog astronauts were also asked to perform some physical activity, in particular during this mission, the crew medical officer established a minimum of 1 h per day. In the habitat, there was a treadmill, an exercise bike and some soft mats for exercises. The subjects were free to choose the kind of activity they preferred, aiming to remain under anaerobic threshold, with a target heart rate of 140–160 bpm. The crew was asked to face emergencies and other unexpected situations during the mission, notified by the mission control in response to real solar activity for example or common emergencies in regular space missions (e.g., air leak, fire, solar storm). Facing simulated emergencies often created highly stressful situations for the crew members that were forced to abandon their working schedule and to rethink the mission plan after spending hours in solving the scenarios. Meals were consumed together, as the crew agreed, to improve social contact between the analog astronauts, following a balanced diet proposed by a nutritionist. Water intake was high, in fact, the crew members were encouraged to drink since in stressing conditions dehydration could be detrimental for performance. No caffeine, alcohol or sweet beverages were allowed, the subjects could drink just water or warm herbal infusions.

During the experiment week a large amount of data were recorded. Every day at 8 a.m., the subjects measured their temperature, body weight, blood pressure, heart rate, arterial pressure, SpO2.

Wearable devices (Mi Smart Band 6, Xiaomi, China) were used to monitor daily step count, physical activity, heart rate during physical exercise and at rest, and sleep parameters. Algorithms from the builders app were used in the data analysis since we had no access to raw data from the devices. Each day crew medical officer obtained saliva and urine samples. Saliva samples have been collected immediately after awakening, before breakfast (Fig. 1).

Fig. 1

Schema of experimental protocol using saliva and urine samples from T0 to T6 (T0: the first day of the mission and T6 the last day of the mission) in young analog astronauts

Ethical considerations

This study was conducted following the Helsinki Declaration and was approved by the Ethical Committee of the Health Authorities of the Province of Padua approved the study (practice number 3843/AO/16). All the volunteers signed an informed consent. The experiment has been conducted in the Analog Astronaut Training Center (AATC) in Poland in October 2021.

Sample collection

Biofluid was collected every day from T0, the first day of the mission, to T6, the last day of the mission. Saliva samples were collected, about 1 mL, using a Salivette device (Sarstedt, Nümbrecht, Germany), centrifuged at 3000 rpm for 20 min (Mrakic-Sposta et al. 2020; Giacon et al. 2022; Bosco et al. 2023; Brizzolari et al. 2023), aliquoted and stored at − 80 °C until assayed and thawed only once before analysis. Also, urine samples were collected by voluntary voiding in a sterile container, aliquoted and stored at − 20 °C until assayed and thawed only once before analysis.

Biomarkers assessment in salivaReactive oxygen species (ROS)

Electron paramagnetic resonance spectroscopy (EPR), 9.3 GHz, X-band, (E-Scan, Bruker Co., MA, USA) was used to detect ROS production, at 37 °C using a Temperature Controller unit (Noxigen Science Transfer & Diagnostics GmbH, Germany), interfaced with the spectrometer. Methods were previously described (Mrakic-Sposta et al. 2020; Giacon et al. 2022; Bosco et al. 2023; Brizzolari et al. 2023), briefly CHM spin probe (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine) was used for ROS production detection, and a stable radical CP· (3-carboxy2,2,5,5-tetramethyl-1-pyrrolidi-nyloxy) was used as an external reference to convert ROS determinations into absolute quantitative values (μmol min−1). Spectra acquired were recorded and analyzed using Win EPR software (version 2.11) standardly supplied by Bruker.

Total antioxidant capacity (TAC)

The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity assay, by a widely used kit-based commercial method (No. 709001, Cayman Chemical, Ann Arbor, MI, USA), was used in saliva samples. The method was previously described (Vezzoli et al. 2016).

Cortisol

The concentration of free cortisol in the saliva was quantitatively determined through ELISA method according to the protocol of the manufacturer's kit (COR(Cortisol) ELISA Kit; FineTest, Wuhan Fine Biotech Co.) as previously described (Giacon et al. 2022).

Leptin and IGF-1

Leptin and IGF-1 levels were measured in saliva by means of enzyme immunoassay (ELISA) kits (cat. No. EH0216; FineTest, Wuhan, China) and (cat. No.EH0165; FineTest, Wuhan, China) respectively. The methods were previously described (Micarelli et al. 2022, 2023).

Biomarkers assessment in urine8-isoprostane (8-iso-PGF2α)

Lipid peroxidation was assessed by immunoassay of 8-isoprostane concentration (Cayman Chemical, Ann Arbor, MI, USA) in urine. Samples and standard were read at a wavelength of 512 nm. The results were normalized by the urine creatinine values. The method was previously described (Bosco et al. 2018a, b, 2023; Giacon et al. 2022; Mrakic-Sposta et al. 2019, 2020, 2022; Vezzoli et al. 2023).

Interleukin-6 (IL-6)

IL-6 urinary levels were determined by ELISA kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The method was previously described (Mrakic-Sposta et al. 2015, 2020; Giacon et al. 2022).

All the samples and standards were read by a microplate reader spectrophotometer (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). The determinations were assessed in duplicate, and the inter-assay coefficient of variation was in the range indicated by the manufacturer.

Creatinine, neopterin and uric acid

Urinary creatinine, neopterin, and uric acid concentrations were measured by isocratic high-pressure liquid chromatography (HPLC) method, as previously described (Dellanoce et al. 2014; Vezzoli et al. 2016) over the range of 0.125–1 μmol/L, 0.625–20 mmol/L, and 1.25–10 mmol/L for neopterin, uric acid, and creatinine levels, respectively. Inter-assay and intra-assay coefficients of variation were < 5%. Methods were previously described (Glantzounis et al. 2005; Mrakic-Sposta et al. 2019; Giacon et al. 2022).

Physical exercise

Daily physical exercise was strongly encouraged. Subjects could freely choose to either run on a treadmill (Urbogym V620MS, Urbogym, Poland) or cycling on an exercise bike (SportPlus sp rb 950 i.e., sportplus, Hamburg, Germany). Normal fitness exercises such as push-ups and crunches and muscles stretching were recommended before and after the main exercise. Distance and duration of the effort were controlled both on the fitness device itself and through the wearable device (Mi Smart Band 6, Xiaomi, China) which also monitored heart rate, daily step count and esteemed caloric expenditure.

Sleep

Sleep monitoring was obtained through both a normal chronometer to monitor the duration of sleep and to further investigate sleep phases and sleep duration we utilized data from the wearable devices (Mi Smart Band 6, Xiaomi, China). Sleep quality was self-reported by the subjects following a 1 (worst) to 10 (best) Visual Analogue Scale (VAS). Sleep duration was determined by mission control, outside of the habitat, and it was shifted from normal light cycle. Sleep debt increased during the week. The crew slept in a small room, in bunk beds, without switching off artificial light.