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. Author manuscript; available in PMC: 2024 Sep 20.
Published in final edited form as: Neuroprotection. 2024 Jun 4;2(3):228–242. doi: 10.1002/nep3.47

Tolerability and first hints for potential efficacy of motor-cognitive training under inspiratory hypoxia in health and neuropsychiatric disorders: A translational viewpoint

Svea-Solveig Mennen 1,2,#, Maren Franta 1,2,#, Martin Begemann 1,2, Justus B H Wilke 1, Roman Schröder 1,2, Umer Javed Butt 1, Jonathan-Alexis Cortés-Silva 1, Umut Çakır 1,2, Marie Güra 1,2, Markus de Marées 3, Vinicius Daguano Gastaldi 1,2, Johannes Burtscher 4, Julie Schanz 5, Matthias Bohn 6, Martin Burtscher 7, Andreas Fischer 5, Luise Poustka 8, Peter Hammermann 9, Markus Stadler 10, Fred Lühder 11, Manvendra Singh 1, Klaus-Armin Nave 12, Kamilla Woznica Miskowiak 13, Hannelore Ehrenreich 1,2,14,✉
PMCID: PMC7616549  EMSID: EMS196792  PMID: 39404697

Abstract

Hypoxia is more and more perceived as pivotal physiological driving force, allowing cells in the brain and elsewhere to acclimate to lowered oxygen (O2), and abridged metabolism. The mediating transcription program is induced by inspiratory hypoxia but also by intensive motor-cognitive tasks, provoking a relative decrease in O2 in relation to the acutely augmented requirement. We termed this fundamental, demand-dependent drop in O2 availability “functional hypoxia.” Major players in the hypoxia response are hypoxia-inducible factors (HIFs) and associated prolyl-hydroxylases. HIFs are transcription factors, stabilized by low O2 accessibility, and control expression of a multitude of genes. Changes in oxygen, however, can also be sensed via other pathways, among them the thiol-oxidase (2-aminoethanethiol) dioxygenase. Considering the far-reaching biological response to hypoxia, hitherto mostly observed in rodents, we initiated a translational project, combining mild to moderate inspiratory with functional hypoxia. We had identified this combination earlier to benefit motor-cognitive attainment in mice. A total of 20 subjects were included: 13 healthy individuals and 7 patients with depression and/or autism spectrum disorder. Here, we show that motor-cognitive training under inspiratory hypoxia (12% O2) for 3.5 h daily over 3 weeks is optimally tolerated. We present first signals of beneficial effects on general well-being, cognitive performance, physical fitness and psychopathology. Erythropoietin in serum increases under hypoxia and flow cytometry analysis of blood reveals several immune cell types to be mildly modulated by hypoxia. To obtain reliable information regarding the “add-on” value of inspiratory on top of functional hypoxia, induced by motor-cognitive training, a single-blind study—with versus without inspiratory hypoxia—is essential and outlined here.

Keywords: brain, cognition, erythropoietin, functional hypoxia, high-parameter flow cytometry, immune cells, oxygen saturation, physical fitness, plasticity

1. Preface

Hypoxia is increasingly recognized as an important physiological mediator. A specific transcriptional program, induced by a reduction in oxygen availability, for instance, inspiratory hypobaric hypoxia at high altitude, allows cells in the brain and elsewhere to adapt to lowered oxygen and reduced energy metabolism. This transcriptional program is partly independent of Lee et al.1 and Jain et al.2 and partly controlled by hypoxia-inducible factors (HIFs) together with the associated prolyl-hydroxylases, which act as oxygen sensors. Changes in oxygen, however, can also be sensed via other pathways, by alterations in metabolite levels or the generation of reactive oxygen species by mitochondria. HIFs are transcription factors that bind to hypoxia-responsive elements to modulate the expression of innumerable genes.3–7 We note that the same transcription program in brain, also HIF-controlled, is induced by intensive motor-cognitive tasks, which provoke a relative reduction in oxygen availability in relation to the acutely increased requirement.

2. Functional Hypoxia

We coined the term “functional hypoxia” for this fundamental, demand-dependent decrease in oxygen availability.8–11 Functional hypoxia seems to be of critical importance for the adaptation to higher physiological need, providing substantial “brain hardware upgrade” (i.e., more dendritic spines and more new neurons). This brain hardware upgrade, in turn, underlies the subsequently improved brain performance. Erythropoietin (EPO), upregulated by hypoxia via HIF, likely plays a decisive role in these processes, which can be imitated by treatment with recombinant human EPO.8

3. HIF/Prolyl-Hydroxylases and ADO, a New Player

Next to HIF/prolyl-hydroxylases, there are other mediators of the hypoxia-induced transcription program. Interestingly, the team around Sir Peter Ratcliffe, who in 2019, together with WG Kaelin and GL Semenza, obtained the Nobel Prize for his work on cellular reactions to hypoxia, identified only recently another exciting, hypoxia-responsive enzyme, namely, a thiol-oxidase, called cysteamin (2-aminoethanethiol) dioxygenase (in brief ADO).12 ADO modifies N-Cys residues to achieve oxygen-regulated, proteolytic degradation of signal molecules. The fact that ADO acts directly on the protein stability of these molecules, indicates a much faster hypoxia response as compared with HIF/prolyl-hydroxylases. ADO regulates, for instance, RGS4 and RGS5 (regulators of G-protein signaling) N-degron substrates, modulates G-protein-coupled calcium signals and mitogen-activated protein kinase (MAPK). On the other hand, RGS4 and RGS5 are HIF-inducible transcripts. This leads to the conclusion that in the hypoxia reaction, both systems, ADO and HIF, act closely and temporally shifted together. ADO is ubiquitously found in the body, but seems to be expressed at particularly high levels in brain.13

There is as yet not much literature available regarding ADO and hypoxia. In particular, there is no study published that investigated the consequences of a lack of ADO (using knockout mice) on basic and complex behavior or cognitive proficiency. This knowledge, however, is indispensable for understanding the different elements of the hypoxia response and for assessing their therapeutic potential, as well as of the highest relevance for translation to humans. Therefore, we are now, in collaboration with Sir Peter Ratcliffe, addressing these questions.

The here presented results of this exploratory pilot trial allow us to design a single-blind study on motor-cognitive training under normoxia versus hypoxia, including healthy participants and neuropsychiatric patients.14,15 We plan in this single-blind study to determine ADO expression in peripheral blood mono-nuclear cells (PBMCs) upon normoxia versus hypoxia. In a first screening of ADO expression, using our single cell transcriptome data of the murine hippocampus, we found ADO downregulated upon hypoxia in most cell types (except oligodendrocytes; data not shown). This could well be an (in)direct negative feedback loop of the activated enzyme on its transcript. Since ADO activity is oxygen dependent, we will include in the PBMC expression analyses ADO target molecules, for example, RGS4, RGS5, or MAPK.

Conclusions regarding brain functions in this experimental human study are only possible through neuro-psychological testing and evaluation of motor-cognitive performance, whereas in ADO knockout mice, brain tissue can be histologically and biochemically investigated, too. Since ADO modulates G-protein-coupled receptors/pathways, we expect in ADO knockout mice a disturbed neurotransmitter profile and/or function under hypoxia and perhaps even under normoxia. This in turn should be unmasked by abnormal behavior compared with wildtype littermates, and permit first deductions concerning the physiological role of ADO.

4. Clinical Pilot Study to Explore Tolerability and First Evidence of Efficacy of a Defined Motor-Cognitive Training Program Under Inspiratory Hypoxia

Considering the above sketched hypoxia response and its vital physiological effects, thus far mostly observed in rodent experiments, we initiated a translational project. This project should combine inspiratory and functional hypoxia which we had found to be of major benefit for motor-cognitive accomplishment in mice.11

The respective exploratory clinical pilot study commenced in June 2023 and was completed in December 2023. It included healthy subjects and first patients with major depression and/or autism spectrum disorder (ASD) to investigate tolerability and first hints of potential efficacy regarding cognitive performance and physical fitness. The study acronym in the granted ethical proposal (Ethic Committee of the Georg-August-University, Göttingen; study number 36/3/23) is HYPOX-ADULT.

The study was supposed to provide the basis for exploiting possible benefits of moderate, normobaric, inspiratory plus functional hypoxia for healthy subjects and the treatment of individuals with neuropsychiatric disease.14 The underlying premise is that 3.5 h daily of motor-cognitive training over 3 weeks under normobaric hypoxia will lead to enduring improvement in cognition and physical fitness. The hypoxia-regulated brain EPO system is likely among the crucial mediators of this improvement.8

Hypoxia training starts each day at 16% O2 (“landing on the Zugspitze”) and within about 1 h, the chamber reaches 12% O2 (“Mount Blanc plateau”)—in contrast to normoxia (21% O2). The hypoxia chamber and all equipment for motor-cognitive training are presented in Figure 1A–D. The highly stable oxygen concentration curve in the chamber (comprising all days of all trials) provides an excellent quality control (Figure 2). In addition, range of room temperature and humidity are given in the blue inset. Continuous CO2 monitoring revealed that values remained overall below 1%.

Figure 1. Hypoxia chamber.

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(A) Panorama view of the hypoxia chamber with devices for motor-cognitive training: Ergometer, treadmill with large video monitor (Kinomap®-App) for entertainment (both medical devices from h/p/cosmos sports & medical GmbH, Nussdorf/Traunstein, Germany), and 3 work stations for cognition training (HAPPYneuron®, Humansmatter, Lyon, France). (B) Panorama view from the opposite side exhibiting the investigator room outside the chamber. Note the observer window (1.25 m2), which allows to always spot the whole chamber. (C) View from the investigator desk through the observer window. Two of four laptops allow monitoring of all data (Datico Sport & Health GmbH, Burghausen, Germany) and two following cognitive performance of subjects online (HAPPYneuron®). (D) Machine room in the basement (maintained by Hoehenbalance & Technical Control Board).

Figure 2.

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Quality measures of the chamber. Presented is the oxygen concentration of all trials over all days. Due to transient technical problems with data recording in one trial, the respective days were excluded. Note the highly accurate conditions. Inset: Mean values of humidity and temperature, showing rather small standard deviations (SD).

So far, we included—after written informed consent—a total of 22 subjects, 18 men and 4 women, with a mean age of 26.95 ± 7 years. Two male individuals dropped out prematurely, both independent of hypoxia (one because of a flu-like infection, the other for personal reasons). We could assess 7 (35%) patients and 13 (65%) healthy individuals (Figure 3A). Of the seven patients, six had major depression and three were diagnosed with ASD (two with double diagnosis). In the sociodemographic overview, also profession of subjects and training state before inclusion16 are presented. The average time of education of all subjects amounts to >15 years. The multiple-choice vocabulary test (Mehrfachwahl-Wortschatz Test) as a valid and short test to estimate premorbid (here: “pre-experimental”) intelligence resulted in a mean ± SD intelligence quotient of 108 ± 11.17,18 The experimental design of this study and the Göttingen Hypoxia Score are outlined in Figure 3B. In parallel to this study, our collaborators in Denmark, Kamilla Miskowiak and her team, work on a very similar protocol with healthy probands and patients with depression or bipolar disease.19 This corresponding format has enabled us to exchange experience as well as information all along, and it will allow future appraisals over two centers.14

Figure 3. Basic study information.

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(A) Sociodemographic data of participants and (B) overview of the study design, including items of the Göttingen Hypoxia Score. FACS, fluorescence-activated cell sorting; PBMC, peripheral blood mononuclear cells; TTE, time to exhaustion.

We determined the mean course of oxygen saturation of all 20 participants over all training days using pulse oximeters (finger-tip; no application of a vasodilator cream; bluetooth-linked; recording every second; Berry Electronic Tech Co., Ltd.). Considering the remarkably different initial training states among subjects (Figure 3A) and to allow an informative overview, we present the mean oxygen saturation with confidence intervals of participants during their physical resting phases (when partakers mostly sit quietly at a cognition workstation and execute HAPPYneuron). Due to a technical problem, data recording in an early trial (three subjects), was not well synchronized. Therefore, these three subjects had to be excluded here. Exemplified are for N = 17 participants day 4 (initial hypoxia training after 3 days of adaptation) and day 20 (last experimental day). The dashed black horizontal line denotes 75% oxygen saturation and enables the reader to directly visualize the adaptation to hypoxia. Comparing three defined time intervals per training day between days 4 and 20 results in significant increases in oxygen saturation (p = 0.005, p < 0.04, p = 0.001) over the 3-week training (Figure 4). These increases are intuitively illustrated by density plots of oxygen saturation over the three time intervals on days 4 and 20 (Figure 5A). The mean oxygen saturation per day for all 17 participants demonstrates an overall increase and, thereby, an effective adaptation to hypoxia (p = 0.0001; Figure 5B).

Figure 4.

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Oxygen saturation curves of participants during phases without physical training. Shown are the mean saturation curves of N = 17 participants from days 4 (A) and 20 (B), together with the respective confidence intervals during phases without physical training (n = 17 due to transient technical problems with data recording in one trial = three subjects). Note the adaptation to hypoxia. The dotted black line represents 75% oxygen saturation; red “00” signifies the short toilet break at 11.00 h; the orange lines denote the three time intervals employed for comparison of saturation on days 4 and 20; the respective p values are presented on day 20. The comparison between days 4 and 20 for each time interval was done using the average oxygen saturation measured in 5-min intervals. This time interval was chosen to allow biological variation through the evaluated period, but at the same time, account for possible missing data due to the measurement device. Artifacts were filtered out and excluded from the analysis. Statistical analysis was conducted using the Wilcoxon signed-rank test with continuity correction after assessing normality with Shapiro–Wilk.

Figure 5. Density plots of oxygen saturation and mean O2 saturation per day.

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(A) Density plots over the three defined time intervals on days 4 and 20, illustrating the increasing shift over time. (B) Mean oxygen saturation per day over the whole study and N = 17 subjects. Note the continuously ascending slope; t test for the slope coefficient (β1).

Blood of timepoints t1–t4 was collected within the hypoxia chamber immediately before exiting (t0 outside the chamber, directly before entering on the first day; Figure 3B). Again, physiological adjustments are apparent in the course of EPO levels in serum over the 3 hypoxia training weeks, with peak values at timepoint 2 (after 1 week) and a slight decline afterwards (Figure 6A). The sequence of individual EPO values (normalized to individual baseline = 100%; Figure 6B) shows that most participants increase their EPO expression. In contrast, erythrocyte and hematocrit data reveal a remarkable scatter but not even tendencies of an increase, likely because of the intermittent pattern of exposure to moderate hypoxia (Figure 6C,D). For all these parameters, healthy control and patient values appear quite similarly distributed. We note that to avoid the acute effects of hyper- and hypohydration, blood samples were taken under standardized conditions. The heatmap illustrates the relative EPO concentrations, normalized separately for each individual to the mean value of all five timepoints. This presentation settles that most subjects augment EPO in serum with individual peak values mainly at t2 and t3 (Figure 6E).

Figure 6. EPO levels during motor-cognitive training under inspiratory hypoxia.

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(A) Erythropoietin (EPO) serum levels determined by IMMULITE/IMMULITE 1000 EPO. (a solid-phase, enzyme-labeled chemiluminescent sequential immunometric assay), show an increase, starting at timepoint t1 and peaking at t2. Friedman rank sum test over all timepoints. (B) EPO course in the serum of individual participants (normalized to individual baseline at t0 = 100%) reveals that most participants increase EPO expression. (C) Erythrocyte and (D) hematocrit values, measured in the same blood samples (clinical routine testing), scatter but do not reveal changes. Mean ± SEM; N = 18; black dots: healthy subjects; blue dots: patients. (E) The heatmap illustrates the relative levels of EPO, normalized to the mean value of all five timepoints separately for each individual. This plot includes 13 healthy subjects and five patients (rows). While the dark red color denotes a higher level of EPO in relation to this particular individual's mean, the light blue color corresponds to a decline. SEM, standard error of the mean.

Hypoxia, exercise, and physical training at (simulated) high altitude have been associated with changes in the blood transcriptome, immune cell composition, and immune function.20–26 Hence, we determined acute and chronic effects of our intermittent hypoxia intervention on seven major mononuclear immune cell types and 20 minor immune cell subsets. Blood of all timepoints (t0–t4; Figure 3B) was examined by high-parameter flow cytometry (FACSymphony S6; BD) within 1 h (Figures 7 and 8). A single acute (3.5 h) exposure to hypoxia neither affected immune cell composition (Figure 8) nor the expression of the early activation marker CD6927 in major lymphocyte subsets (data not shown). Furthermore, weekly monitoring of the immune cell composition did not reveal any adverse immunological alteration (all parameters within mean ± 1 SD of pre-hypoxia values). However, we found a mild but significant regulation of five immune cell populations by chronic hypoxia, namely, naïve and central memory CD4 + T-cells, total and naïve B cells, as well as total natural killer cells (Figures 7D and 8).

Figure 7. High-parameter immunophenotyping of seven major and 20 minor immune cell subsets in human peripheral blood.

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(A) Major mononuclear cell clusters in human peripheral blood after clean-up gating, downsampling, concatenation, dimensionality reduction using optsne, and PhenoGraph clustering28 in Flowjo (v.10.9.0). (B) Heatmaps depicting side-scatter area (SSC-A) and surface expression of major lineage markers. (C) Uniform gating of major immune cell types into 20 distinct minor immune cell subsets on the cleaned, concatenated, and clustered mononuclear cell data. For quantification see Figure 8. (D) Immune cell subsets significantly regulated by chronic intermittent hypoxia exposure. Effects of chronic hypoxia on immune cell composition were tested depending on data distribution with one-way repeated measure ANOVA and Friedman rank sum test from the ez (Package ‘ez’ version. 4.4-0 [2016]).29 and stats packages in R 4.3.1 (R Core Team. R Foundation for Statistical Computing; 2023). ANOVA, analysis of variance; NK, natural killer.

Figure 8.

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Frequency of major and minor immune cell subsets in human peripheral blood upon acute and chronic exposure to moderate hypoxia. The figure-table shows 20 minor immune cell subsets, normalized to the respective seven major immune cell types. Nomenclature was based on distinct surface marker combinations.30–37 EDTA blood of timepoints t0–t4 (Figure 3B) was collected (t1–t4 within the hypoxia chamber), processed, and subjected to high-parameter flow cytometry (FACSymphony S6; BD) within 1 h after hypoxia. For each participant, a fresh staining master mix was prepared at t0 and used throughout t1–t4. Due to the aberrantly high number of artifacts (>10%) as compared with the other samples (average of 1.6% artifacts), three samples were excluded. One participant was excluded from the analysis owed to a lack of CD45RA negative T-cells and one was excluded due to poor baseline sample quality (>10% artifacts). Statistical analyses were performed using R 4.3.1. Data displayed as mean ± SD; t test = paired two-sided t test; Wilcox = Wilcoxon signed-rank test with continuity correction; RM-ANOVA = repeated measure ANOVA; Friedman = Friedman rank sum test. Methodological details on the flow cytometry procedure are available from the authors upon request. ANOVA, analysis of variance; EDTA, ethylenediamine tetraacetic acid.

Rating the own general well-being on a scale from 0 to 10 is part of the self-rating using the Göttingen Hypoxia Score. We find clear improvement (p = 0.008) when comparing the values of all subjects immediately before the first day in the chamber and after leaving the chamber on the last day (Figure 9A). Regarding the general well-being of patients only (N = 7), the difference after/before shows at least a tendency (p = 0.09). Subjects' grading of their hypoxia side effects according to the Göttingen Hypoxia Score is presented as mean delta values for all 18 days and all symptoms (values after stepping out of the chamber minus before entering) in Figure 9B. The various symptoms seem to go up and down quite irregularly, with a trend of overall reduction towards the end of the 3-week training period. This pattern permits to calculate on a scale from 0 to 3 for each subject the mean value of the difference after versus before for each item over 18 days (Figure 9C). To conclude, symptoms are overall mild and also reveal some adaptation.

Figure 9. Tolerability of motor-cognitive training under inspiratory hypoxia.

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(A) Self-rating of general well-being on a scale of 0–10: The improvement from the first day before entering to the last day after leaving the chamber reaches statistical significance; paired Wilcoxon signed-rank test with continuity correction; N = 20 (cave: partially overlapping circles). (B) Course of the individual symptoms of the Göttingen Hypoxia Score (mean difference of all days after minus before hypoxia presented) shows an irregular pattern, and towards the end of the 3 weeks underscores a gradual adaptation to hypoxia. (C) Symptoms according to the Göttingen Hypoxia Score on a scale of 0–3: Means of the difference of all individual values of all participants for all training days immediately after leaving the chamber versus the values immediately before entering the chamber are given. Note the overall mild symptoms. N = 20; black dots, healthy subjects; red dots, patients.

During exposure to hypoxia over the 3 motor-cognitive training weeks, probands spent a considerable time at the cognition workstation. In fact, the average time per day amounts to 1.83 ± 0.4 h with a mean number of HAPPYneuron tasks/day of 58.83 ± 12.3, resulting in a total of 1059 ± 220 tasks over the 3 weeks. Importantly, the featured tasks are progressively difficult to accomplish in true cognitive training. Figure 10 signifies the stepwise increase in performance in six cognitive domains from week 1 to 2 to 3. With each step, the data scatter expands more and more, nicely reflecting that with increasing difficulty, the field of individual participants is dissociating. This was to be expected. Nevertheless, Jonckheere-Terpstra Trend Tests are highly significant for all domains. In most domains, patients (red dots) show a similar distribution as healthy subjects (black dots).

Figure 10.

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Performance in all six domains of the HAPPYneuron cognitive training. Shown is the mean performance/week in six cognitive domains over the 3 weeks of motor-cognitive training. Performance in each test was calculated by dividing the achieved performance by the maximum achievable performance (100%) in this particular test and multiplying the result with the corresponding level of test difficulty given by HAPPYneuron; Jonckheere Terpstra Trend tests; N = 20; mean ± SEM presented; black dots, healthy subjects; red dots, patients. SEM, standard error of the mean.

Neuropsychological testing before and after the 3 training weeks shows tendencies of improvement in many of the 12 conducted tests. Since subjects differ with respect to the individual selection of improved cognition readouts, we formed a cognition composite score out of all 12 tests.38–41 As shown in Figure 11A, the composite score, calculated based on all data of all subjects before and after the 3 motor-cognitive training weeks under inspiratory hypoxia, includes the cognitive domains attention, visual and verbal learning and memory, reversal learning, working memory, and executive function. It results in Cronbach's alpha of 0.75 (Figure 11B), proving high internal consistency of the intercorrelation matrix and thus authorizing to apply the score. Next, comparing composite score values before and after the 3 weeks, yields a distinct enhancement (Figure 11C). As some limitation, however, it should be noted that the present test package does not entirely exclude practice effects,42 which is particularly evident for the Wisconsin Card Sorting Test (WCST).43 This will have to be considered in future studies with WCST being replaced by another, better-suited test, for example, the WAIS-IV matrix test (deductive reasoning/problem solving). Similar to cognition, the first clear signals of an augmentation of physical fitness upon 3 weeks of motor-cognitive training are seen, with time to exhaustion (TTE) and VO2max44 increasing, the latter obtained using the German version of the Bruce protocol (Figure 12A,B). Evaluating the seven patients only shows positive trends for cognition (p = 0.156) and physical fitness (TTE p = 0.05; VO2max p = 0.08). As described in the literature,45 we also find first indications of a decrease in systolic as well as diastolic blood pressure, particularly in individuals with pre-existing hypertension (data not shown).

Figure 11. Cognitive fitness before and after 3 motor-cognitive training weeks under inspiratory hypoxia.

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(A) Raw data of test performance in the different instruments,46–54 z-standardized. Note that in most cases—even though per se often not significant—mean values are higher after the 3-week hypoxia training. The large standard deviation reflects the interindividual differences in the various cognitive domains; mean ± SEM presented. (B) The conditions reported in (A) are the perfect indication for forming a cognition composite score38–41 which can be used for quantifying overall cognitive enhancement, if Cronbach's alpha, as quality measure of internal consistency of the intercorrelation matrix, exceeds 0.6. (C) On the basis of the very good Cronbach's alpha of 0.75, the cognition composite score was calculated before versus after the hypoxia training for estimating overall cognitive improvement; N = 20; mean ± SEM; paired Wilcoxon signed-rank test with continuity correction. BZT, Buchstaben-Zahlen-Test; RMIE, Reading Mind in the Eyes; SEM, standard error of the mean; TAP-A/-DA/-VS, Testbatterie zur Aufmerksamkeitsprüfung-Alertness/-Divided Attention/-Visual Scanning; TMT-A, Trail-Making Test A; TMT-B, Trail-Making Test B; VF, Verbal Fluency; VLMT, Verbaler Lern-und Merkfähigkeitstest; WCST, Wisconsin Card Sorting Test.

Figure 12. Physical fitness before and after 3 motor-cognitive training weeks under inspiratory hypoxia.

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(A) Time to exhaustion (s) (TTE) is defined as maximum training duration in the standardized ergometer step load test. (B) VO2max = 12 ∗ Wmax + 300 (mL/min), with Wmax being the maximum power level in the standardized ergometer step load test. (A, B) Exhaustion is defined as a subjective feeling of maximum exertion of the test person. Percentage change from individual baseline (=100%) shown; N = 19 (one subject could not be retested due to an acute knee problem acquired independently of the hypoxia training); student's paired t test (TTE); Wilcoxon signed-rank test with continuity correction (VO2max).

The Brief Symptom Inventory55 was given to all 20 subjects (healthy and neuropsychiatrically ill) before and after the hypoxia training phase to check for potential alterations in any of the nine syndrome categories that partly (in the lower scale range) reflect personality traits. Surprisingly, the values for the items depression, anxiety, and obsession–compulsion decreased, whereas those for paranoid ideation, interpersonal sensitivity, and hostility just showed a tendency of reduction. Psychoticism, phobic anxiety, and somatization remained unchanged (Figure 13A). Two established specific instruments, the Beck Depression Inventory (self-assessment instrument)56 and the Hamilton Depression Rating Scale (HAMD-17; external assessment instrument)57 were employed for patients before and after the training weeks and demonstrate improved psychopathology (Figure 13B).

Figure 13. Personality features and psychopathology before and after 3 motor-cognitive training weeks under inspiratory hypoxia.

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(A) Brief Symptom Inventory (BSI): Presented are the nine scales of the BSI. Scale values (0–4) were calculated by totaling the corresponding values for all items and dividing the resulting sum by the number of items; mean ± SEM; N = 20; paired Wilcoxon signed-rank test with continuity correction. Psychopathology measures of depression in patients: Beck Depression Inventory (BDI; self-assessment instrument) and Hamilton Depression Rating Scale (HAMD-17; external assessment instrument) after the 3-week training are expressed as percent individual baseline (=100%); N = 6; paired Wilcoxon signed-rank test with continuity correction. SEM, standard error of the mean.

5. Discussion and Deductions from this Pilot Trial

The present manuscript provides preliminary findings of an open label trial which served to explore mainly tolerability, but also potential signals for efficacy of motor-cognitive training under inspiratory hypoxia.14 We report that this procedure is very well tolerated, reveals signs of adaptation to hypoxia and first indications of benefit for general well-being, cognitive and physical fitness, as well as psychopathology.

The design of the study was strict, straightforward and set up in a way that it can now serve as an ideal blueprint for the upcoming single-blind study. Despite being a pilot trial, we feel that early sharing of all important details and our experience will help others to initiate their own studies. This the more so since evidence emerges for functional hypoxia as a candidate treatment for brain disorders, which thus far have limited therapeutic options. Whether the additional inspiratory hypoxia acts as synergistic or additive momentum on cognition and physical fitness cannot be reliably answered at this time. It requires as an essential next step the single-blind study, comparing motor-cognitive training under normoxia versus hypoxia.

An important additional impetus that we observed in this study, but did not quantify yet, is the relative weight of the social setting (three people training at once).

Most training groups in this trial soon developed a positive, stimulating, and encouraging interaction, which seemed to benefit even patients with ASD or subjects with autistic traits. Furthermore, the “organized” conditions of the study appear to provide valuable hold and structure. This would mean that both, social and structured circumstances, belong to the active factors of this arrangement.

In a recent systematic review, we summarized findings and methodological quality of work investigating hypoxia (10%–16% O2) for ≥14 days in humans as well as the neurobiological mechanisms triggered by hypoxia in rodents.58 Overall, for the majority of studies, the quality turned out to be quite low. Moreover, the comparability of human studies is problematic because of a huge heterogeneity of protocols and mostly very small numbers of participants. While 75% of human studies indicated cognitive and/or neurological benefits, all studies were evaluated as having a high risk of bias. As a cautious conclusion of this systematic review, low-dose intermittent or continuous hypoxia, repeated for 30–240 min sessions, preferably in combination with motor-cognitive training, seems to produce beneficial effects.58 Nevertheless, larger and methodologically stronger translational studies are definitely warranted.

6. Waiting List of Study Participants

The present waiting list of potential participants, both healthy and ill, is fortunately long. Apparently, increasing word-of-mouth advertising as well as distributing information about the trial among patients and self-help groups, in sports clubs, recreational centers, and so forth worked well in this particular setting. At the beginning, we hardly found women who volunteered to participate in the 3-week hypoxia training, leading to an imbalance of genders (Figure 3A). Also, regarding gender distribution, the waiting list is now more promising.

7. Design of the Single-Blind Study Based on this Pilot Trial

Founded on the outcomes of and experience with the pilot study,14 we composed a rational and realistic design of the single-blind study (Figure 14). This study will follow the same experimental protocol as the present one (Figure 3B), just the time spent at the various training stations will be firmly controlled, and subjects will be unaware of exposure to hypoxia versus normoxia (single-blind study). Moreover, follow-up exams (e.g., 2 months after hypoxia training) will be performed to test whether and how long the benefits are lasting. We plan to keep participants “always busy” during the 3.5 h in the chamber. Only during the first 2 days, they are free to rest—at least in between HAPPYneuron sessions—and asked not to start physical exercise before the second day. From then on, they should train just moderately to not overextend themselves prematurely. This is strictly controlled by the observers outside the chamber, who can communicate with subjects in the chamber via walkie-talkie (Motorola Solutions Germany GmbH), and enables a gradual adaptation to hypoxia training.

Figure 14. Design of the next step: Single-blind study on motor-cognitive training under normoxia versus inspiratory hypoxia.

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(A) Mean physical training duration on treadmill. (B) Mean physical training duration on ergometer. (C) Mean velocity on treadmill (km/h). (D) Mean power on ergometer (Watt). (E) Design of the structured motor-cognitive training based on (A) and (B) as predictors of realistic timetables regarding the different workstations. Schedules are given for three subjects training simultaneously and rotating through the three different schedules.

(C) and (D) are left to the participants' own decision allowing an adjustment to the pre-existing training state. Note the relatively homogeneous distribution of parameter mean values in (A)–(D) over the 17 training days.

Regarding the time distribution at the various work-stations, we roughly oriented ourselves on the mean voluntary periods of all individuals at the treadmill, ergometer (Figure 14A,B), and HAPPYneuron (nearly 2 h/day). To not overstrain subjects (with their expectedly different training state at the beginning of the trial, see Figure 3A), we decided not to influence their own selection of training intensity, that is, mean velocity on the treadmill (km/h; Figure 14C) and mean power on the ergometer (Watt; Figure 14D). Nevertheless, we and the co-training individuals in the chamber will encourage them to increase their training efficiency. Figure 14E shows the plan for the slot distribution of three subjects who train simultaneously, starting on day 3. This ensures that the respective workstations are free. The distribution rotates over 3 days and then starts again for each individual with his/her first schedule. Altogether, subjects train in this fashion for 16 days (20 days minus 2 starting days and minus 2 Sundays), meaning that they undergo their schedule five times each and finally execute one additional day with their starting schedule. In case that subjects who trained in normoxia show less benefit compared with the hypoxia group, we will offer them the chance to undergo the hypoxia training earliest after 3 months. Should this offer be welcomed by many test subjects, then later, a single-blinded randomized crossover trial might be an approach worth pursuing.

8. Conclusions

Motor-cognitive training under inspiratory hypoxia (12% O2) for daily 3.5 h over 3 weeks is perfectly tolerated and seems to benefit general well-being, cognitive and physical fitness, as well as psychopathology. EPO serum levels rise under hypoxia and flow cytometry analysis of blood reveals 5 of 20 immune cell types to be slightly regulated by hypoxia. The single-blind study with versus without inspiratory hypoxia will provide information on the “add-on” value of mild to moderate inspiratory on top of functional hypoxia.

Highlights.

  • Motor-cognitive training under inspiratory hypoxia (12% O2) for 3.5 h daily over 3 weeks is very well tolerated by healthy individuals as well as by patients with depression and/or autism spectrum disorder.

  • We find first promising signals of beneficial effects on general well-being, cognitive performance, physical fitness, and psychopathology.

  • Our protocol of motor-cognitive training under inspiratory hypoxia does not adversely affect the mononuclear cell composition in the blood of adults.

  • To obtain reliable information regarding the “add-on” value of mild to moderate inspiratory on top of functional hypoxia, induced by motor-cognitive training, the planned single-blind study—with versus without inspiratory hypoxia—is required.

Acknowledgments

This work has been supported by the European Research Council (ERC) Advanced Grant to HE under the European Union's Horizon Europe research and innovation program (acronym BREPOCI; Grant Agreement No. 101054369), as well as an ERC Consolidator Grant to Kamilla Woznica Miskowiak (acronym ALTIBRAIN; Grant Agreement No. 101043416), in collaboration with Hannelore Ehrenreich. Furthermore, the study has been supported by the Max Planck Society and the Max Planck Förderstiftung. Research in the labs of Hannelore Ehrenreich and Klaus-Armin Nave is funded by DFG TRR-274/1 2020-408885537. Klaus-Armin Nave is supported by the Adelson Medical Research Foundation.

Funding information

ERC Advanced Grant acronym BREPOCI, Grant/Award Number: 101054369; ALTIBRAIN, Grant/Award Number: 101043416; DFG, Grant/Award Number: TRR-274/1 2020-408885537

Footnotes

Author Contributions

Concept, design, and supervision: Hannelore Ehrenreich, Kamilla Woznica Miskowiak, and Klaus-Armin Nave. Funding acquisition: Hannelore Ehrenreich, Kamilla Woznica Miskowiak, Klaus-Armin Nave, and Peter Hammermann. Drafting manuscript and display items: Hannelore Ehrenreich, Svea-Solveig Mennen, and Maren Franta. Data acquisition/generation: Svea-Solveig Mennen, Maren Franta, Martin Begemann, Jonathan-Alexis Cortés-Silva, Justus B. H. Wilke, Roman Schröder, Marie Güra, Julie Schanz, and Andreas Fischer. Data analyses and interpretation: Maren Franta, Svea-Solveig Mennen, Jonathan-Alexis Cortés-Silva, Roman Schröder, Justus B. H. Wilke, Manvendra Singh, Umut Çakır, Fred Lühder, and Hannelore Ehrenreich. Literature searches: Hannelore Ehrenreich, Svea-Solveig Mennen, Maren Franta, Justus B. H. Wilke, Martin Burtscher, and Umer Javed Butt. Continuous input/supporting data management: Umer Javed Butt, Umut Çakır Johannes Burtscher, Markus de Marées, Matthias Bohn, Vinicius Daguano Gastaldi, Markus Stadler, Peter Hammermann, Luise Poustka, and Fred Lühder. All authors read and approved the final version of the manuscript.

Conflict of Interest Statement

The authors declare no conflict of interest.

Ethics Statement

The study acronym in the granted ethical proposal (Ethic Committee of the Georg-August-University, Göttingen; study number 36/3/23) is HYPOX-ADULT.

Data Availability Statement

All original data are available from the authors upon request.

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Associated Data

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Data Availability Statement

All original data are available from the authors upon request.

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