Orbital spaceflight onboard the Space Shuttle and the International Space Station (ISS) were operated in a normoxic, sea-level atmosphere, i.e., 14.7 psi and 21% oxygen. Spacewalks, or extravehicular activities (EVAs) are required to be performed in the lower pressure, mildly hyperoxic atmosphere, of a spacesuit. Typically, the EVA suit atmosphere was 4.3 psi and >95% oxygen. To avoid decompression sickness, the EVA required a denitrogenation (oxygen prebreathe) protocol that took several hours (Brady and Polk, 2011).

Future deep space exploration missions, particularly those on planetary surfaces, will entail a higher frequency of EVAs than on missions to low Earth orbit. Also, it is not desirous to require a prolonged oxygen prebreathe prior to the EVA. Instead, quick exit out of the habitat or pressurized rover would be beneficial, to enable frequent EVAs during exploration missions. The current concept of operations under consideration by the Artemis Program is an ambient vehicle/habitat ‘exploration atmosphere’ of 8.2 psi and 34% O2 (piO2 128 mmHg) (Norcross et al., 2013). This would allow for a shorter oxygen prebreathe (∼20 min), greatly shortening the logistics before exiting the vehicle for an EVA and greatly facilitating exploration operations. EVA suit atmosphere would still be hyperoxic (piO2 222 mmHg). Deep space explorers would therefore be frequently cycling between hypoxic and hyperoxic conditions. There is evidence that hyperoxia exposures influenced brain perfusion, cognition, and EEG activity (Damato et al., 2020; Damato et al., 2022b). There is very little existing research data regarding the effects of such atmospheric cycling on physiology.

Spaceflight mission stressors other than altered atmosphere, such as microgravity, circadian misalignment and physiologic stress, result in the persistent dysregulation of the human immune system (Krittanawong et al., 2022; Rooney et al., 2019). The phenomenon is characterized by reductions in T and NK cell function, altered cytokine profiles, and the reactivation of latent herpesviruses (Crucian et al., 2015; Bigley, 2019; Crucian et al., 2014; Mehta et al., 2017; Rooney et al., 2019). While these alterations have not caused widespread clinical issues, some crewmembers experience immune-related adverse events, including manifestations of symptomatic herpes viral reactivation, allergy, and respiratory distress (Crucian et al., 2016a IJGM). Three case reports have been published of astronauts regarding clinically relevant immune compromise, and dermatitis on orbit, two of which confirmed the involvement of latent herpesvirus reactivation (Crucian et al., 2016b; Mehta et al., 2022; Mehta et al., 2024). One ISS crewmember experienced zoster (laboratory confirmed by skin swab) on orbit, and the outbreak was correlated with peaks in both immune dysfunction and stress hormone concentrations (Mehta et al., 2024). Another case correlated dermatitis flares with operational stressors (Crucian et al., 2016a). In a third case, virus ‘evolution’ onboard ISS, defined as increasing micro-variants, was documented (Mehta et al., 2022). Such cases are important as they demonstrate that the immune dysregulation in astronauts can progress to operationally-impactful adverse clinical events during flight. It is reasonable to hypothesize that the immune dysregulation observed aboard ISS missions will intensify during longer missions in deep space, thereby placing crewmembers at elevated clinical risk (Crucian and Sams, 2009).

Other environments, such as saturation diving and Antarctic winterover, share many isolation and mission stressors with spaceflight, including station lifestyle, extreme environment, and 6–12 months deployments. Saturation diving can also include frequent changes to atmospheric pressure, extended duration living in altered atmospheric conditions, and challenges with humidity and sea life induced contamination (Brubakk et al., 2014). Findings from immune studies at Concordia Antarctic Station were surprisingly discordant from spaceflight findings. Whereas spaceflight generally results in a suppression of leukocyte function, at Concordia (innate) cellular function was generally sensitized, with functional responses elevated (Feuerecker et al., 2014; Feuerecker et al., 2019). The only confounding variable thought best to be responsible for this sensitization was the persistent hypobaric hypoxia as Antarctica is a mountain, and Concordia Station sits at 10,606 feet in elevation (piO2 ∼97 mmHg).

Hypoxia is known to regulate and/or influence immunity, inflammation, and physiology in a generally stimulatory fashion, as reviewed by Taylor et al. (Taylor and Colgan, 2017; Damato et al., 2022a). Hypoxia can influence immune cellular functional capacity (e.g., proliferation, development), or can function primarily via transcriptional changes mediated by hypoxia-inducible factor. While there is variability among altitude/hypoxia studies, generally, effects on immunity are stimulatory, and pro-inflammatory (Kubo et al., 1998; Hartmann et al., 2000; Coussons-Read et al., 2002; Facco et al., 2005; Ermolao et al., 2009), while other reports suggest lymphocytosis, or HPA axis mediated immune suppression (Siqués et al., 2007; Chen et al., 2012; Oliver et al., 2013; Kleessen et al., 2005) or innate cellular migration and inflammation (Pahl et al., 2006). Hypoxia also specifically influences anti-tumor immunity (Arnaiz and Harris, 2022). Physical activity or exercise at altitude in a hypoxic environment had differential influences (de Aquino Lemos et al., 2013; Mazzeo, 2005; Tiollier et al., 2005; Choukèr et al., 2005). A recent study suggests that normobaric hypoxia training and increased erythropoiesis impacts iron metabolism, which can challenge maintenance of immune function (Nolte et al., 2025). The effects of hypoxia on immunity appear highly biased, meaning some aspects of inflammation/immunity may be suppressed, whereas others may be heightened or sensitized. This effect may be exploited as a treatment for specific disease states related to an alteration in immune bias. For example, hypoxia can be beneficial for asthma allergy, or eczema, which are all considered to be ‘T_H2’ diseases, or certain tumors resulting in intra-tumor localized hypoxia (Schultze-Werninghaus, 2008; Engst and Vocks, 2000; Hatfield et al., 2015).

Current denitrogenation prebreathe protocols performed prior to conducting a spacewalk on the ISS, from a normoxic vehicle to a hypobaric spacesuit, last about 4 h. EVAs will be much more frequent during upcoming planetary exploration. To optimize time spent exploring the Lunar surface and minimize prebreathe durations while maintaining a safe risk posture for decompression sickness risk prevention, an ‘exploration atmosphere’ is being planned for the Artemis program, where the vehicle will be at 8.2 psi and 34% oxygen (or similar), allowing for significantly shorter prebreathe durations prior to EVA decompression. There are varied literature reporting immune dysregulation associated with hypoxia, primarily derived from studies of people living at altitude, as well as decompression stress.

Immune dysregulation has also been well documented to be associated with other spaceflight stressors, such as microgravity, isolation, circadian misalignment, or altered diet. This has been observed in studies of astronauts during flight, as an amalgamation of all these mission stressors, and also in ground-space-analog studies, where such variables may be studied individually. Given that deep space missions will likely be conducted in a uniquely hypobaric/hypoxic environment with intermittent hypobaric/mildly hyperoxic EVAs, the current study was conducted in a hypobaric chamber housed at the Johnson Space Center. Subjects lived in the chamber for 11 days in a mildly hypoxic environment, and during several mission days participated in hyperoxic simulated ‘EVAs’. This operational scenario is a high-fidelity representation of the atmosphere planned for deep space missions. Biosampling was conducted on several days, both before and after EVA. The objective was to allow a determination of the effects of both hypoxia and hyperoxia (and cycling therein) on immunity, inflammation, and latent herpesvirus reactivation.

There are reports that hypoxia may have benefit as a prophylactic, or therapeutic, for some disease states, and that the addition of intermittent hyperoxia during the exposures may heighten the magnitude of benefit (Uzun et al., 2023). A mechanism of action may be the upregulation of reactive oxygen species and hypoxia-inducible genes. Other reports suggest that hyperoxia exposures are well tolerated (Netzer et al., 2024; Walker et al., 2018). There is, however, no broad evidence base informative of the effect of routine cycling between hypoxia and hyperoxia on aspects physiology. The current study was conducted at the Johnson Space Center, to assess the condition of living in a habitat at mild hypoxia, with periodic decompression with hyperoxic EVA activity simulation (atmospheres being defined by vehicle and mission constraints), on multiple aspects of physiology. The goal was to ascertain if the atmospheric cycling increased any specific clinical risks. The immune assessment was part of that parent study, and utilized a battery of assays previously validated to monitor multiple aspects of immune dysregulation observed in astronauts during spaceflight. Blood samples were collected as described in the schedule above, timed to specifically assess the effect of mild hypoxia, hyperoxic EVA activity, decompression stress, and alternating between the two environments. These assays include peripheral leukocyte distribution as a measure of in-vivo mobilization of immune subsets generally related to immune responses, plasma cytokine concentrations as an indicator of the in-vivo status of the hormonal regulation of immunity between particular immune ‘biases’, and immune cell functional capacity assessed following mitogenic stimulation. Additionally, saliva stress hormone concentrations and the subclinical reactivation of latent herpesviruses were monitored daily.

The data revealed minimal alterations, immune, stress, etc., resulting from the mildly hypoxic cabin environment, primarily associated with the MD3 a.m. sampling (Table 1). At this point, the subjects had experienced almost 3 continuous days in the exploration atmosphere environment. On the surface, this would seem somewhat discordant with our previous finding of profound immune sensitization and latent virus reactivation while living at Concordia Station, Antarctica. These results, as they themselves were somewhat polar opposite from the spaceflight findings, were generally attributed to the confounding variable of persistent hypobaric hypoxia. However, it is relevant that the hypoxia experienced at Concordia Station is profound, with an atmosphere pp O2 of 101.6 mmHg (with an ambient pressure of 522 mmHg), as compared to 159 mmHg at sea level (760 mmHg ambient). This chamber study was NOT designed to assess impactful hypoxia, and was only adjusted to 141–144 mmHg pp O2 but in a significantly lower pressure than Concordia (422/493 mmHg ambient), to coincide with the planned deep space atmosphere. The authors believe this is why noteworthy effects of hypoxia, despite a more pronounced hypobaric stress, were not observed in this chamber study.

The post-hyperoxic EVA findings however, primarily MD3 and MD7 p.m. samplings, were consistent. A redistribution of leukocytes was evident, primarily an increase in neutrophils, with a relative decrease in lymphocytes, monocytes, basophils and eosinophils (Table 2). The RBC, hematocrit and hemoglobin were all increased, and RBC morphology was altered, with an increase in corpuscular volume and hemoglobin (Table 2). Lymphocyte subsets were also altered post EVA, with an increase in the T cell percentage, a reduction in cytotoxic T cells (without a corresponding increase in helper T cells), and an increase in the maturation state of the helper T cell subsets, but a converse reduction in the maturation state of the cytotoxic compartment (Table 2). B cell subsets also moved towards a increased maturation state. Plasma cytokine dysregulation was also evident post- EVA, with an increase in the concentration of several cytokines associated with inflammation (IL-6, IL-12, IL-18) and chemokines also associated with inflammation (MCP-1, IP-10) (Table 3). By MD7, IL-1ra was also increased, confirming a pro-inflammatory state in the subjects. Alterations were also observed for several other cytokines, including RANTES, PDGF, and others, that likely point to disruption of other immunoregulatory processes (Table 3). Minimal alterations in plasma cytokines were observed by MD10, indicating that the previous alterations were indeed associated only with the hyperoxic EVA exposures and not the cabin hypoxia. Leukocyte function, as determined by supernatant cytokine production following blood leukocyte mitogenic stimulation in culture (48 h) was dramatically altered post EVA exposure (Table 4). Increases in multiple cytokines were observed after the EVA activities on MD3 and MD7, with far less alterations at the ‘cycling’ timepoints on the mornings of MD7 and MD10. These results confirm a ‘sensitization’ or increase in immune function was evident, moreso seemingly in the innate populations (LPS stimulation) and broadly (PMA-I stimulation), than was observed in following the T cell specific stimulation (anti-3/28) (Table 4).

The primary finding from this study is that, while living in a mildly hypoxic exploration atmosphere did not have a profound impact on immunity, the participation in hypobaric/hyperoxic EVA activities impacted numerous and varied immune parameters. A summation of the primary significant impacts of hyperoxic EVA are represented in Figure 4. This study was designed to be an applied, clinical research assessment, that monitored four basic categories of outputs: (A) leukocyte distribution, (B) plasma cytokine concentrations, (C) leukocyte function, and (D) latent virus reactivation. We have previously discussed how these parameters generally provide a good snapshot of immune mobilization (A), immunological response biases (B), environmental impacts on function (C), and an adverse clinical outcome that may be quantitated (D). This is insufficient for a detailed biological pathway analysis, however is sufficient for a broad assessment of subject immune function, determining the nature of any dysregulation, and projections of clinical risk. The significant findings confirm immune mobilization of primarily innate cells, a generally pro-inflammatory sensitization, and profound environmental impacts in reducing leukocyte function, without a concurrent rise in latent virus reactivation as is commonly observed in astronauts (Figure 4). Most of the cytokines that were increased in the subject’s plasma, although not to a profound level, are indeed associated with inflammation. The reductions in cell function were most remarkable in both the consistency across the 13 cytokines measures, and across the 3 distinct stimuli. PMA and ionomycin stimulates the nucleus directly and bypass the intracellular signaling pathways required for activation in-vivo, yet it yielded the most consistent reduction in function (11/12 cytokines). In previous stress-analog studies a reduction in cellular function is almost always associated with both stress and the reactivation of latent herpesviruses. The most likely reason for the absence of latent virus reactivation in this study, and the unchanging cortisol levels, seems to be the relatively short duration of the chamber habitation, and the intermittent ‘rest’ days allowing a degree of recovery, something that does not happen during spaceflight of any duration.

It should be noted that although stimulations during cell culture were utilized to assess immunocyte functional capacity, these cultures occurred during normoxic and normobaric laboratory conditions. An excellent indicator of direct hypoxia/hyperoxia effects on cell function would be inclusion of altered-atmosphere cell culture. In such a study, alterations may be ‘induced’ by the altered atmosphere culture conditions, even in healthy individuals, essentially creating an artifactual dysregulation that is not representative of the in-vivo condition. That was not the goal of this study. Instead, the goal was to assess dysregulation induced in-vivo, by performing ‘normal’ stimulations ex-vivo and observing functional decrements. This is analogous to spaceflight investigations of astronauts with cellular function altered by microgravity. Dysregulation may be induced in healthy subjects’ cells by culturing in modeled-microgravity conditions, in a bioreactor or clinostat device. For functional assessment of astronauts during spaceflight however, blood samples are collected onboard ISS, and returned for terrestrial ‘static’ (normal, 1xG) cell culture to monitor function. While altered culture conditions are informative for basic cell biology studies, to assess an individual’s immune function in a clinical fashion is not desirous to induce artifactual decrements by culturing in unusual conditions.

Although the changes described above associated with hyperoxic simulated EVAs are noteworthy, they are generally associated with mobilization, inflammation, and cell sensitization. We are therefore unsurprised that in only an 11 days study, there was no increase in stress hormones or any clinically relevant reactivation of latent herpesviruses. Such reactivation, as is commonly observed during spaceflight, is generally associated with immune suppression, and is commonly observed in astronauts during spaceflight (Rooney et al., 2019). Immune suppression was not observed during this study. Nor does this study represent the creation of a true ground-based Space Flight analog in terms of immune response. The goal was not to recreate mission stressors but to create a high-fidelity situation to assess the effect of altered atmospheres in parallel with the physical activity of EVA.

The results from this study confirm previous observations that altered oxygen partial pressures in the atmosphere can profoundly influence immunity. Limitations may include an imperfect analog relevance to spaceflight, as other mission associated stressor (microgravity, radiation, etc.) are absent. Generally, the literature regarding hypoxia exposure is derived from assessments of individuals living at altitude, and the findings generally indicate that such hypoxia exposure is immune sensitizing (Kubo et al., 1998; Hartmann et al., 2000; Coussons-Read et al., 2002; Facco et al., 2005; Ermolao et al., 2009). The previous study at Concordia station confirmed this scenario but using a more sophisticated panel of assays as defined by immune alterations in astronauts (Feuerecker et al., 2014; Feuerecker et al., 2019). The effect of very mild hypoxia in conjunction with intermittent hyperoxic EVA on immunity was unknown. Decompression stress is also known to trigger an inflammatory response. Particularly following hyperbaric (vs hypobaric) exposures, decompression also causes elevation of inflammatory markers even without clinical signs or symptoms of decompression illness (Mitchell, 2024; Mrakic-Sposta et al., 2024). Although similar studies in hypobaric exposures are sparse, blood markers for hypobaric exposures show elevation of inflammatory markers (Connolly et al., 2023). The data seem to indicate that the mild hypoxia condition, even less than hypoxia experienced in an equivalent altitude of Denver, is not disruptive. The hyperoxia and decompression stress of EVAs result in a profound mobilization inflammation and sensitization of immunity as clearly evident in the samples taken after the simulated EVAs (PM). Humans during deep space missions will be a complicated applied experiment consisting of multiple mission stressors and influencing variables, including microgravity stress circadian misalignment, altered diet, and all this in a much more limited habitable volume with communication delays. Altered atmosphere simply adds yet another variable that can influence human physiology. We suggest further characterization is warranted as scenarios with different atmospheres are tested with more EVAs, as well as an ability to monitor immunity and crew members during deep space missions. Having a better understanding of immune changes during atmospheric cycling will allow for development of broad biomedical countermeasures capable of restoring immune function.