Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity - A Randomized Controlled Cross-Over Trial
Phase: NA
Conditions: Oxygen Toxicity, Oxidative Stress, Hyperoxia, Hyperbaric Oxygen, Healthy Subjects (HS), Diving Medicine, Hydrogen-oxygen Gas
Sponsor: Blekinge Institute of Technology

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
1 
 
 
RESEARCH PLAN 
Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity:  
A Randomized Controlled Cross-Over Trial
 
 
 
 
Unique Protocol-ID: BTH-6.1.1-0165-2025  
 
NCT-Number: NCT-ID not yet assigned  
 
Document date: 2025-10-27 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
2 
 
 
1.1. PROJECT TITLE: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity: 
- A Randomized Controlled Cross-Over Trial. 
 
1.2. SWEDISH TITLE: Effekt av vätgas på hyperbar syrgastoxicitet:  
- En randomiserad kontrollerad studie. 
 
2.2 INVESTIGATING RESEARCH FACILITIES 
 
1. Blekinge Institute of Technology (BTH) - Marine System Engineering  
2. Swedish Armed Forces (FM DNC) - Diving and Naval Medicine Centre in Karlskrona. 
3. Blekinge Hospital (BLS) Karlskrona, Department of Pulmonary Medicine 
4. Lund University, Department of Respiratory Medicine, Allergology, and Palliative Medicine 
5. Lund University, Dept. of Translational Medicine 
6. University of Gothenburg - Sahlgrenska Academy, Occupational and Environmental Medicine, 
Department of Public Health and Community Medicine 
7. Karolinska Institutet (KI), Department of Physiology and Pharmacology 
8. Karolinska University Hospital, Perioperative Medicine and Intensive Care 
9. Blekinge Institute of Technology (BTH) – Institute of Health/THIA 
10. Sahlgrenska University Hospital, Gothenburg, Department of Anaesthesia and Intensive care 
 
 3. ESTIMATED DURATION OF THE STUDY 
Approximate study period of 5 years (2025 – 2030). The duration of the study for each participant is 
four days in total. 
 
4. ABSTRACT (SYNOPSIS) 
Introduction: Professional divers within the Swedish Armed Forces are frequently exposed to 
extreme environments where the human body is not fully adapted. To mitigate diving-related risks 
such as decompression sickness (DCS), caused by the accumulation of inert nitrogen bubbles, 
oxygen-enriched breathing gases are commonly used by both military and technical divers. Despite its 
benefits, the use of oxygen-enriched breathing gases is limited by the risk of adverse effects from 
increased partial pressures of oxygen (PO₂) and the increased formation of reactive oxygen species 
(ROS). The maximum allowable PO₂ is defined by the risk of CNS-toxicity, while exposure duration 
is primarily limited by pulmonary oxygen toxicity (POT)(1). Current limits for exposure are primarily 
based on reductions in lung vital capacity (VC)(2), although other markers may better reflect 
oxidative stress. Despite strict regulations, there is currently no established method to counteract the 
oxidative damage during prolonged oxygen exposure. Recent studies suggest that the already known 
diving gas, hydrogen, has antioxidative properties, neutralizing cytotoxic oxygen radicals and 
providing potential cellular protection.  
 
Aim: We aim to investigate if a diving gas with a small fraction of hydrogen could reduce Pulmonary 
Oxygen Toxicity. 1-2% fraction of hydrogen to an oxygen-enriched breathing gas (FO₂ = 0.98-0.99, 
FH₂ = 0.02-0.01) reduce Pulmonary Oxygen Toxicity (POT) in divers exposed to a partial pressure of 
1.75 ATA for 240 minutes. In parallel, the study aims to elucidate the underlying pathophysiological 
mechanisms associated with POT. 
 
Materials and methods: The study will follow a double-blind, randomized, crossover design with two 
exposure sessions, 1-2% of hydrogen (intervention) and 1-2% of nitrogen (control) to oxygen 
breathing gas (FO₂ = 0.98-0.99) in divers exposed to a partial pressure of 1.75 ATA for 240 minutes.  
These exposures will be separated by a minimum of 2 weeks. Each session will be followed by a post-
exposure assessment day. Pulmonary Functions Tests (PFTs) and biochemical markers of oxidative 
stress and of neuronal injury will be measured before and after each intervention. With each 
participant undergoing both the intervention (H2-group) and control (N2-group) they will serve as their 
own control for comparative analysis.  
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
3 
 
 
Participants: Recruitment will be conducted via email inquiries, targeting active professional divers 
(N=32) from the Swedish Armed Forces. 
 
Statistical Analysis: Baseline characteristics will be summarized using descriptive statistics. Within-
participant differences in FVC, will be analyzed using paired t-tests, while categorical variables will 
be assessed with Wilcoxon matched-pairs signed-rank tests, as well as using regression models. 
Statistical significance is set at a two-tailed p-value < 0.05.  
 
Significance: If successful, the findings of our study could contribute to the development of safer 
diving protocols and optimize operational efficiency while minimizing oxygen toxicity risks.  
Keywords:  
Diving; Hyperoxia; Ventilation; Oxygen Toxicity; Pulmonary Oxygen Toxicity; Oxidative Stress;  
 
 
Abbreviation list: 
ATA = Atmosphere absolute 
ATS = American Thoracic Society  
BIBS = Built-In Breathing System  
BLS = Blekinge Hospital Karlskrona  
BTH = Blekinge Institute of Technology  
CCRs = Closed-Circuit rebreathers  
DAN = Divers Alert Network 
DCS = Decompression Sickness  
DLCO = Diffusion Lung Capacity for Carbon Monoxide 
EANx = Enriched Air Nitrox 
ELF= Epithelial Lining Fluid  
FeNO =  
FM DNC = Swedish Armed Forced Diving and Naval Medicine Center 
FVC = Forced Vital Capacity 
Heliox = Gas mixture with oxygen and helium 
HBOT = Hyperbaric Oxygen Therapy 
HPNS = High-pressure Nervous Syndrome 
iOS= Index of [Oxygen Stress] 
Nitrox = Oxygen-enriched gas with nitrogen 
NOAA = National Oceanic and Atmospheric Administration 
PExA = Particle in Exhaled Air  
PEFR = Peak Expiratory Flow Rate  
PFTs = Pulmonary Function Tests  
POT = Pulmonary Oxygen Toxicity  
ROS = Reactive Oxygen Species  
SOB = Shortness of Breath 
SVC = Slow Vital Capacity 
TLC = Total Lung Capacity 
TLV = Total Lung Volume  
VC = Vital Capacity 
RV = Residual Volume 
FRC = Functional Residual Capacity 
5.  AIM, OBJECTIVES AND OUTCOMES 
Aim: 
To investigate whether a high-oxygen diving breathing gas enriched with a small fraction of hydrogen 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
4 
 
 
(1–2%) can reduce pulmonary oxygen toxicity (POT) in divers. In parallel, the study aims to elucidate 
the underlying pathophysiological mechanisms associated with POT. 
Objectives: 
1. To characterize the oxidative stress response in the lungs during hyperoxic exposure to diving 
breathing gases by means of pulmonary function tests and molecular analysis of exhaled breath. 
2. To characterize systemic oxidative stress during hyperoxic exposure to diving breathing gases 
through the measurement of circulating and urinary biomarkers. 
3. To determine whether the addition of hydrogen to a high-oxygen diving breathing gas confers 
measurable protective effects against POT, as assessed by lung function, exhaled breath particels, and 
systemic biomarkers. 
4. To identify predictive markers associated with individual susceptibility or resistance to oxygen-
induced damage and the potential efficacy of hydrogen-enriched diving breathing gases. 
5. To evaluate the effect of breathing hyperbaric oxygen, with and without addition of hydrogen, on 
the central nervous system.  
Exposure: 
Participants will be exposed to two different high-oxygen diving breathing gas mixtures at a partial 
oxygen pressure of 1.75 ATA for 240 minutes: 
• 
Intervention gas: Fraction of oxygen (FO₂) = 0.98–0.99; fraction of hydrogen (FH₂) = 0.01–
0.02 
• 
Control gas: FO₂ = 0.98–0.99; fraction of nitrogen (FN₂) = 0.01–0.02 
This study design allows for a direct comparison between the physiological effects of hydrogen- and 
nitrogen-enriched diving breathing gases under hyperbaric conditions. 
Primary Outcome 
• 
The primary outcome will be the difference in the absolute change in forced vital capacity 
(ΔFVC) between the two diving breathing gas exposures. 
• 
ΔFVC is defined as the individual change from pre-exposure to post-exposure values within 
each condition. Main comparison will be the mean ΔFVC following hydrogen-enriched gas 
exposure versus control gas exposure, reflecting the physiological impact of hydrogen on 
pulmonary oxygen toxicity. 
Secondary Outcomes: 
• 
Changes in diffusing capacity for carbon monoxide (DLCO) 
• 
Changes in exhaled particle concentration and composition measured using Particle Exhaled 
Air (PExA) analysis 
• 
Changes in fractional exhaled nitric oxide (FeNO), as a marker of airway inflammation 
• 
Changes in the Index of Oxygen Stress (iOS) 
• 
Biomarkes in blood and urine.  
• 
Changes in concentrations of biomarkers of neuronal injury in blood 
___________________________________________________________________________ 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
5 
 
 
6. BACKGROUND AND RATIONALE  
 
The choice of breathing gas mixture is a crucial factor in optimizing diver safety and reducing 
physiological risks associated with high pressure exposure in underwater environments. Among the 
most well-documented risks in diving medicine are the physiological challenges arising from 
accumulation of inert gases, leading to conditions such as nitrogen narcosis and decompression sickness 
(DCS) (3) While nitrogen narcosis intensifies with increasing partial pressure, the risk of DCS is 
determined by a combination of factors. These include the duration of hyperbaric exposure, ascent rate, 
physical exertion and breathing gas composition. One strategy to reduce the accumulation of inert gas 
is to increase the fraction of oxygen (O2). This principle forms the basis for the use of alternative gas 
mixtures such as pure oxygen, Nitrox (Enriched Air Nitrox, EANx) and Heliox (helium and oxygen) 
(4), each optimized for specific depth ranges and physiological demands (5).  
 
However, the use of oxygen-enriched breathing gas mixtures is limited by the risk of oxygen toxicity. 
Under high pressure conditions where the partial pressure of oxygen (pO₂) exceeds established safety 
thresholds (6,7). In such environments, the elevated pO₂ significantly increase the formation of reactive 
oxygen species (ROS), contributing to oxidative stress. Despite technological advancements in gas 
composition, oxygen toxicity remains a critical limiting factor, restricting both depth and duration in 
diving operations.  
At sea level, where the partial pressure of oxygen is approximately 0.21 atmospheres absolute (ATA), 
natural defense mechanisms within the body, are generally sufficient to mitigate oxidative stress by 
neutralizing ROS, thereby effectively preventing cellular injury that leads to tissue damage. However, 
when oxygen is administrated at partial pressures exceeding 0.6 ATA, prolonged exposure may 
overwhelm these endogenous defense mechanisms (8,9). Organs particularly susceptible to oxygen-
induced damage include the lungs and the central nervous system, leading to conditions collectively 
known as pulmonary oxygen toxicity (POT) and central nervous system (CNS) toxicity. 
To mitigate the risks of acute toxicity, professional divers strictly adhere to oxygen exposure limits 
regulated by various safety guidelines over the past few decades  Traditionally, the most widely used 
parameter for assessing POT has been a reduction in vital capacity (VC) (10) Although researchers 
have tried to find better biomarkers for POT, but no useful correlations have yet been found (11). 
However, some physiological measurements have been found that may reflect pulmonary oxidative 
damage better than VC (12). Tailoring safety measures based on these measurements may represent a 
significant advancement in the optimization of exposure guidelines for professional divers. 
 
There is currently no established method to directly mitigate the formation of ROS and associated 
oxidative toxicity encountered during diving. However, emerging research suggests that hydrogen 
possesses antioxidative properties through its ability to selectively neutralize cytotoxic oxygen 
radicals, thereby offering potential cell-protective effects (13–16). 
Although the antioxidative role of hydrogen in counteracting POT remains a subject for investigation, 
its application as an inert gas in diving has already been investigation with high pressures of hydrogen 
without any toxically effect besides narcosis at very high pressures (17) A diver who had previously 
experienced HPNS symptoms under similar conditions while using trimix blend, reported no such 
effects when using a helihydrox gas mixture of 3% O₂, 59% He, 38% H₂. This observation does not 
only support the hypothesis that H₂ may alleviate HPNS, but also contributes to our understanding of 
its narcotic potential, which has only been observed at absolute pressures exceeding 24 ATA, 
corresponding to depths equivalent to 230 meters, way beyond the range of most conventional 
operational dives. (18) 
 
Further historical and experimental data reinforce hydrogen’s physiological promise, the practice of 
using hydrogen as a diluent gas is well-documented in several high-profile research programs, most 
notably those conducted by Compagnie Maritime d’Expertises (COMEX). Being a pioneer in 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
6 
 
 
demonstrating the feasibility of hydrogen-oxygen breathing under extreme hyperbaric conditions. 
Around 100 hydrogen dives were carried out in controlled hyperbaric chamber environments and 
offshore operations. Among these, the Hydra VIII experiment in 1988 reached a simulated depth of 701 
meters, representing one of the deepest hydrogen-based dives ever recorded (19). Parallel efforts by the 
Institute of Marine Research in Norway involved dives with similar breathing gas mixtures to depths 
of 300 meters, while the U.S Navy conducted numerous experimental H₂-dives under chamber 
conditions. Although the exact depth profiles of these U.S. Navy dives remain classified or unpublished, 
they add further weight to the international interest in H₂ hyperbaric applications. (20) Collectively, 
these efforts provide a robust empirical foundation for renewed exploration of hydrogen as a therapeutic 
and operational tool in hyperbaric physiology. (21) 
 
 
7. CLINICAL SIGNIFICANCE 
Even though oxygen-enriched breathing gases have been extensively studied, the potential benefits of 
incorporating small fractions of hydrogen remain largely unexplored. As outlined above, hydrogen-
enriched breathing gas emerges as a promising strategy for mitigating oxidative stress, offering a 
potential means of extending the operational limits of deep diving. Investigating its physiological and 
neurological effects could provide valuable insights into the management of ROS and oxygen toxicity 
under hyperbaric conditions. By emphasizing the need for further research into strategies for 
neutralizing oxidative stress and enhancing diving safety, our intervention aims to explore the 
potential benefits of hydrogen-enriched breathing gas in detail. A deeper understanding of potential 
protective effects of hydrogen could significantly enhance diver safety, expanding the boundaries of 
modern diving medicine. 
 
 
8. MATERIAL AND METHODS:   
 
Study Design:  
This is a double-blind, randomized, crossover trial (Figure 1). Both participants and investigators 
conducting the assessments and analyses will be unaware (blinded) of which treatment is assigned to 
each group during the intervention, to mitigate placebo and confounding factors. 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
7 
 
 
Figure 1. Outline of the trial 
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
8 
 
 
 
8.2 INTERVENTION OF THE TRIAL 
The study is conducted over four days, with each exposure followed by a post-exposure assessment 
the next day. This design minimizes variability and ensure an accurate evaluation of the results in a 
controlled environment. Exposures are divided into two separate sessions, with at least two weeks 
between each crossover visit. However, participants will not be continuously monitored between 
sessions, no additional visits are planned beyond scheduled pre- and post-intervention assessments. 
Screening Visit (week 0): Randomization 
1. Consenting Process (obtained inform of consent) 
2. Screening for inclusion/exclusion criteria  
3. Medical History + previous sAE correlated to diving  
4. Randomization 1:1 to either intervention or control group  
 
Study Visit #1 (week 1) pre-exposure  
à Assessments are carried out prior to each individual intervention, on the same day and 
shortly before the first scheduled intervention in both groups. 
1. Self-reported symptoms (breathlessness, cough and other respiratory symptom[s]) 
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry (FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC) and body plethysmography 
(TLC, RV, FRC) 
5. Diffusing lung capacity for carbon monoxide (DLCO).  
6. PExA, Blood and Urine collection (for later analysis of biomarkers of oxidative stress and neuronal 
injury) 
 
Study Visit #1 (week 1) post-exposure 
àAssessments are carried out 30-120 min after (post exposure) of each individual intervention 
1. Self-reported symptoms  
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry: FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC. Body plethysmography (TLC, 
RV, FRC) 
3. FeNO 
5. DLCO. 
6. Blood and urine collection (for later analysis of biomarkers of oxidative stress and neuronal injury) 
 
Study Visit #2 (week 1) post-exposure 
à Assessments are carried out the following day, approximately 12-36 h post exposure of each 
individual intervention.  
1. Self-reported symptoms  
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry: FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC. Body plethysmography (TLC, 
RV, FRC) 
5. DLCO 
6. PExA, Blood and Urine collection (for later analysis of biomarkers of oxidative stress and neuronal 
injury) 
à Intervention Crossover 1:1  
By implementing a 1:1 crossover design with fixed randomization, equal number of participants are 
allocated to either the intervention or the control group. This ensures that each group receives both 
interventions and effectively allow each participant to serve as their own control. By balancing the 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
9 
 
 
sequence in which participants receive the interventions, potential carryover effects are evenly 
distributed across both groups minimizing the risk of wash-over effects between the phases. (22)  
Study Visit #3 (week 2) pre-exposure 
à Assessments are carried out prior to each individual intervention, on the same day and 
shortly before the second scheduled intervention in both groups. 
1. Self-reported symptom[s] 
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry: FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC. Body plethysmography (TLC, 
RV, FRC) 
5. DLCO 
6. PExA, Blood and Urine collection (for later analysis of biomarkers of oxidative stress and neuronal 
injury) 
 
Study Visit #3 (week 2) post-exposure 
Assessments are carried out 30-120 min after (post exposure) the second individual intervention 
1. Self-reported symptom[s] 
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry: FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC. Body plethysmography (TLC, 
RV, FRC) 
5. DLCO 
6. Blood and urine collection (for later analysis of biomarkers of oxidative stress and neuronal injury) 
 
Study Visit #4 (week 2) post-exposure 
à Assessments are carried out the following day, approximately 12-36 h after (post exposure) of 
the second scheduled individual intervention.  
1. Self-reported symptoms (breathlessness, cough and other respiratory symptom[s]) 
2. Tremoflo - Ocillometry (iOS) 
3. Fractional Exhaled Nitric Oxide (FeNO) 
4. Spirometry: FVC, FEV1, IC, FEF25, FEF50 and FEF75, PEF, SVC. Body plethysmography (TLC, 
RV, FRC) 
5. DLCO 
6. PExA, Blood and Urine collection (for later analysis of biomarkers of oxidative stress and neuronal 
injury) 
 
9. 1 PARTICIPANTS AND RECRUITMENT  
 
Study population:  
Recruitment will be conducted via mail inquiry, targeting (N=32) individuals that are active military 
divers, with no history of serious diving-related injuries or long-term complications. 
Inclusion criteria: 
• 
Military divers actively serving, aged 20 years or older  
• 
Meeting the Swedish Armed Forces physical standards for diving 
Exclusion criteria: 
• 
Ongoing infection or illness that may impact pulmonary function or immune response. 
• 
Use of alcohol or smoking cigarettes within 48 hours, as this may influence oxidative stress 
• 
Diving within 48 hours or oxygen diving within 2 weeks, to minimize potential residual 
effects of the intervention. 
• 
Use of medications that could affect oxidative stress, lung function, or neurological status 
• 
Medical history of serious diving-related injuries or long-term complications 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
10 
 
 
Informed consent: 
Following orally and written information with a detailed explanation of study procedures, potential 
risks and anticipated benefits. Participants will have the opportunity to ask questions. Each participant 
will sign an informed consent before any study related procedures takes place. Subjects may at any 
given time withdraw their consent and leave the study without having to motivate the reason for 
withdrawal. 
 
 
9.2 RANDOMIZATION 
Following screening and inclusion, participants will be randomly assigned to begin with either the 
intervention or the control. A fixed 1:1 allocation scheme will be used to ensure that an equal number 
of participants (N=16) start in each side of the trial. This design minimizes potential washover-effects 
where the first exposure might influence the subsequent phase. After the initial phase, a crossover will 
be implemented, allowing each participant to serve as their own control. Comprehensive pulmonary 
function tests (PFTs), including spirometry, body plethysmography, diffusing capacity for carbon 
monoxide (DLCO), and regional ventilation assessments, will be performed before and after each 
exposure. 
10. BLINDING PROCEDURE: 
To minimize the risk of bias and prevent confounding factors that could lead to placebo effects, a 
double-blinded design is implemented, meaning that both participants and investigators involved in 
outcome assessments remain blinded to group allocation. This approach is essential to ensure that any 
observed physiological effects or symptoms are directly attributed to the intervention rather than 
expectations of the investigator or preconceived beliefs related to the participants.  
Participants: The breathing gas mixtures, consisting of oxygen with or without a low fraction of 
hydrogen, will be administered either via demand regulators during submerged (in-water exposure) or 
via the Built-In Breathing System (BIBS) used in the hyperbaric chamber (HAUX 2300). All delivery 
systems will be identical in external appearance with standardized components to ensure that no 
visual differences could reveal group allocation. Preparation and handling of the gas mixtures will be 
carried out by unblinded technical personnel. These are designated staff members are exclusively 
assigned to gas logistics and are not involved in data collection or outcome evaluation, thereby 
maintaining participant blinding throughout the study. 
Investigators: Those conducting pulmonary function assessments and analysing data will remain 
unaware of which exposure each participant is receiving to ensure objectivity in measurements and 
interpretations. Only personnel who is responsible for the mixing of gas are aware of in which order 
each participant is undergoing the intervention or the control, respectively. By blinding investigators, 
we reduce the risk of “observer bias,” preventing their beliefs or expectations from affecting data 
collection or analysis. This ensures that both investigators and participants remain blinded throughout 
the study, maintaining an unbiased data collection process.  
 
 
11. Hyperbaric Oxygen Dive Protocol:  
Participants will perform wet dives or “dry dives” inside a hyperbaric chamber at an elevated 
atmospheric pressure of 1.75 ATA for 240 minutes, corresponding to the highest approved oxygen 
exposure according to Swedish dive regulations (RMS Dyk 2013). Within the intervention the 
participants are administrated one of the exposure breathing gases.  
 
Risk management:  
Participation in this study entails potential risks related to both medical safety and personal integrity. 
However, the overall risk of harm is assessed to be low. The planned medical assessments are 
predominantly non-invasive and include standard pulmonary function testing, supplemented by urine 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
11 
 
 
collection and venous blood sampling. The latter procedure may, in some cases, cause mild 
discomfort, such as pain or localized hematoma at the site of needle insertion. 
The study involves controlled exposure to hyperbaric oxygen using diving breathing gases at 1.75 
ATA for 240 minutes. During this time, participants will inhale a gas mixture containing 98–99% 
oxygen. Although this exposure is compliant with the Swedish Armed Forces’ current diving 
regulations for oxygen diving (RMS Dyk 2013) and adheres to established safety protocols, it is well 
known that oxygen doses within this range are relatively high and can affect pulmonary function. 
Similar exposures have previously been associated with a transient group-level reduction in vital 
capacity (VC) of approximately 2%. This effect is reversible and has not been linked to long-term 
functional impairment in earlier studies. One of the key scientific objectives of this study is to 
systematically investigate the pathophysiological processes underlying pulmonary oxygen toxicity 
(POT), in order to contribute to more evidence-based and relevant safety limits for future oxygen use 
in hyperbaric environments. 
General diving-related risks include barotrauma to the middle ear, sinuses, or lungs due to inadequate 
pressure equalization. This risk is considered low, as all participants are professional divers trained in 
pressure exposure and hyperbaric safety. Decompression sickness (DCS), typically caused by inert 
gas accumulation, is not relevant in this study. The diving breathing gases contain 98–99% oxygen 
and only 1–2% nitrogen (in the control condition), a level far below what would pose any significant 
risk of DCS. 
The use of hydrogen gas (1–2%) in one of the diving breathing gas mixtures is considered 
physiologically and technically safe. The gas is delivered directly via a breathing regulator (or 
chamber mask), preventing accumulation in free volumes and eliminating ignition risk. Hydrogen has 
previously been used safely in diving breathing gases at substantially higher concentrations, without 
any reported toxic effects. Notably, the Compagnie Maritime d’Expertises (COMEX) conducted 
hydrogen-based dives, both in chambers and during offshore operations. The most extreme project, 
Hydra VIII in 1988, simulated a depth of 701 meters with no reported adverse events. 
The study includes the collection and handling of sensitive personal data, which always carries a 
potential risk to personal privacy. Data handling will be conducted in full compliance with the 
General Data Protection Regulation (GDPR). All data will be pseudonymized, and the associated code 
key stored separately in a secure, access-restricted location. The risk of unauthorized access or data 
misuse is considered low. 
There is also a possibility that participants may be informed of previously unknown health conditions, 
such as unexpected pulmonary findings or reduced oxygen saturation. Any pathological findings will 
be communicated to the participant—regardless of the presence of symptoms—and appropriate 
information about associated risks will be provided. No health-related information will be shared with 
the Swedish Armed Forces or any third party without the participant’s explicit consent. 
Based on operational data from the Swedish Navy, no incidents of central nervous system oxygen 
toxicity (oxygen-induced seizures) have been reported in the past 10 years under diving conditions 
regulated by RMS Dyk 2013. Therefore, the risk of such events is assessed as extremely low. 
Furthermore, the fact that exposure in this study occurs at rest—rather than during physical activity—
further reduces the risk. 
The hydrogen concentration in the diving breathing gas is well below the lower flammability limit in 
oxygen (4%). Thus, the participant is not exposed to any risk of fire or explosion. Although 
theoretical accumulation of hydrogen in a closed space due to leakage from tanks or chamber masks 
could occur, continuous hydrogen leak detection will be implemented to mitigate these already 
minimal risks. Medical preparedness on site will follow the safety protocols in RMS Dyk 2013.  
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
12 
 
 
 
12. OUTCOME MEASUREMENTS: 
 
12.1 Technical devices and medical equipment  
Our study includes a wide range of measurements to evaluate outcomes, many of which require the 
use of specialized technical devices. While certain assessments can be performed using standardized 
instruments commonly used in clinical settings, others specifically demand advanced techniques for 
gas composition analysis, imaging technologies, or laboratory-based biochemical evaluation. By 
integrating multimodal endpoints into the study design, we aim to strengthen our hypothesis and 
capturing both functional and molecular changes associated with POT, as well as the potential 
protective effects of hydrogen supplementation. Following equipment will be used to conduct the 
respective outcome assessments: 
 
Vitalograph™ Devices  
Vitalograph™ (23) is a provider of advanced pulmonary function testing devices designed for clinical 
use and unlike standalone spirometry units, these systems are PC-based and require connection to a 
computer to operate. This setup enables real-time visualization of results and allows analysis to be 
performed via dedicated software. By integrating advanced flow-sensing technology with user-
friendly interfaces, these systems facilitate accurate and efficient lung function assessment in clinical 
environments. Airflow is measured using a Fleisch-type pneumotachograph, which captures laminar 
flow through micro-capillary tubes. Lung volumes are derived through flow integration, with data 
collected at high sampling rates and automatic temperature compensation. This construction ensures 
high precision and long-term stability in accordance with international standards ATS/ERS 2019 and 
ISO 26782:2009 (24) with measurements of following parameters.  
 
Spirometry with body plethysmography:: FVC, FEV₁, FEV₁/FVC ratio, FEF25–75%, IC and PEF. 
TLC (Total Lung Capacity), RV (Residual Volume), and FRC (Functional Residual Capacity). 
→ Device model: Vitalograph™ Pneumotrac or equivalent. 
 
Airway inflammation: Fractional exhaled nitric oxide (FeNO)  
→ Device models: Vitalograph™ FeNO, NIOX VERO™ 1 
 
Oscillometry Testing 
Oscillometry is a non-invasive technique for assessing airway resistance and reactance during tidal 
breathing. Serving as a valuable tool for detecting minor deviations in peripheral airway mechanics. 
Key parameters such as R5, R20 and X5 offer detailed insights into both central and small airway 
function, without the need for forced respiratory maneuvers. Beyond its clinical applications, 
oscillometry is particularly relevant for detecting early-stage toxicological responses and respiratory 
changes associated with inhalation exposures and pharmacological interventions. 
→ Tremoflo™ C-100 (Thorasys) 2 
 
Devices for Diffusing Capacity (DLCO) Testing 
Assessment of pulmonary gas exchange efficiency is essential in both clinical diagnostics and 
respiratory trials, providing a direct measure of alveolar-capillary function. Typically performed using 
single-breath DLCO method, which requires administration of a standardized gas mixture containing 
carbon monoxide (CO) and helium (He), as well as advanced gas analysis to quantify the amount of 
gas diffused across the alveolar-capillary membrane. DLCO values are adjusted for hemoglobin levels 
to improve measurement accuracy. Device solutions employed in this project include: 
→ MGC Diagnostics™ DLCO, Vyaire™ Vmax3 
 
 
1 https://www.niox.com/en/niox-vero/about-niox-vero/  
2 https://www.thorasys.com/solutions/tremoflo-c-100 
3 https://www.vyaire.com/products/vyntus-one-pulmonary-function-system  

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
13 
 
 
→ ndd® EasyOne Pro4  
→ Vitalograph™ COMPACT™ Medical Workstation (DLCO integration)5 
 
PExA (Particle in Exhaled Air)  
The method to sample lining fluid from small airways with the PExA method (Particles in Exhaled 
Air) is a unique, non-invasive technique allowing sampling endogenous aerosol particles from the 
small airways during a special breathing maneuver. It represents a pioneering technology developed 
in Sweden, originating from research at the Department of Occupational and Environmental Medicine 
at the Sahlgrenska Academy, University of Gothenburg. The method enables the collection of 
particles that are formed when the epithelial lining fluid (ELF) burst when small airways open during 
inhalation. The samples can be processed for advanced molecular analysis, including mass 
spectrometry for both proteins and lipids. This provides a novel window into lung biology and is 
particularly valuable for identifying non-invasive biomarkers associated with airway inflammation, 
epithelial integrity, and early toxicological responses. 
 
Since the PExA technique is relatively new, there are currently no established guidelines by European 
or Swedish authorities. By this, the procedure is based on incorporated protocols from peer-reviewed 
research and will strictly follow the manufacturing instructions to ensure consistent and reliable data 
collection.  
The subjects are instructed to perform a standardized breathing maneuver starting with an exhalation 
at normal flow rate to residual volume, breath holding for 5 seconds, followed by a maximal 
inhalation to total lung capacity, immediately followed by a normal exhalation to functional residual 
capacity. Exhalation flow is measured by an ultrasonic flow meter (OEM flow sensor; Spiroson-AS, 
Medical Technologies, Zürich, Switzerland), enabling visualization of the expiratory flow and 
volume. Between breathing manoeuvres, the subject breaths particle-free air tidally for 10 to 30 
seconds. Each sampling session continued until 200 ng of exhaled particles is collected. 
 
12.2 Pulmonary Function Tests (PFTs) 
Following assessments are integrated into the study protocol to provide a comprehensive evaluation of 
lung function and furthermore to determine efficacy of hydrogen supplementation in mitigating POT. 
 
Spirometry and Body plethysmography: 
All subjects will perform forced spirometry from total lung capacity (TLC) to residual volume (RV) 
in triplicate, meaning that three acceptable measurements are obtained. The highest values recorded 
from these acceptable tests will be used for analysis. Following parameters will be measured: 
o 
Forced Vital Capacity (FVC) 
o 
Slow Vital Capacity (SVC) 
o 
Forced Expiratory Volume in one second (FEV₁) 
o 
FEV₁/FVC ratio 
o 
Peak Expiratory Flow Rate (PEFR) 
o 
Total Lung Capacity TLC  
o 
RV, FRC) 
 
Briefly the maneuver is as follows: Spirometry is performed according to the guidelines of the 
European Respiratory Society (ERS) (25).  These guidelines dictate requirements for equipment 
calibration, test execution, and data analysis. In Sweden, national lung function testing guidelines are 
largely aligned with these international standards, ensuring that measurements of FVC, FEV₁, 
FEV₁/FVC ratio, and PEFR are accurate and comparable. 
 
 
4 https://nddmed.com/products/complete-pulmonary-function-machine/easyone-pro/  
5 https://vitalograph.com/products/compact-medical-workstation/ 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
14 
 
 
 
Fractional Exhaled Nitric Oxide (FeNO) 
FeNO will be used as a non-invasive biomarker of airway inflammation and oxidative stress. Nitric 
oxide (NO) is produced endogenously in the respiratory epithelium, primarily via the enzyme 
inducible nitric oxide synthase (iNOS), which is upregulated in response to inflammatory stimuli. 
Hyperoxic conditions, such as those encountered during oxygen-rich diving exposures, are known to 
induce both oxidative stress and secondary inflammatory responses in the pulmonary system. 
Measuring exhaled NO-levels provides a real-time indicator of airway inflammation that contribute to 
evaluation of the antioxidative effects of hydrogen supplementation. 
 
Oscillometry  
Oscillometry serves as a composite measurement designed to quantify the overall impact of oxygen-
induced oxidative stress on lung tissue. This method will be performed in accordance with established 
standards (26) to ensure accurate and reliable measurement of respiratory impedance and resistance. 
 
Briefly the maneuver is as follows:  
Measurements of pulmonary function are taken before and after each dive to assess changes in 
pulmonary function and oxidative stress. This allows us to evaluate the potential mitigating effects of 
hydrogen supplementation on POT. All calculations and interpretations of the oscillometry data will 
strictly follow published methodologies to ensure consistency and reliability in data collection. In 
accordance with these methodologies, the oscillometry technique will be implemented in our study 
using the Tremoflo system, which is known for its ability to provide detailed assessments of small 
airways function—particularly relevant for detecting subtle changes in pulmonary oxidative stress 
levels in this context. 
 
Diffusion Capacity for Carbon Monoxide (DLCO) 
The DLCO test is a standardized test to assess a pulmonary disease, by measuring the ability of the 
lungs to transfer carbon monoxide to the red blood cells in pulmonary capillaries. Carbon monoxide 
(CO) is used due to its 200-fold higher affinity for hemoglobin than oxygen (O₂) 
 
Briefly the maneuver is as follows: Participants will be instructed to stay in a sitting position while 
breathing from a mouthpiece connected to a CO-gas mixture. Participants will first exhale smoothly to 
residual volume (RV) and then take a maximal inhalation to total lung capacity (TLC) of a gas 
mixture (0.3 % CH4, 21% O2, 0.3% CO, balanced with N2). After a 10-second breath-hold, they will 
perform a final exhalation to RV, during which the concentration of CH4 and CO will be measured. 
Alveolar volume (VA), inspired volume (VI), will be calculated. Since hemoglobin concentration 
affects DLCO variability, Hb will be analyzed to minimize errors.  
13.2 Biological sample collection and biochemical analysis 
Blood sampling: 5ml EDTA (Proteinomics, Lipidomics, Metabolomics), 2,5ml PAX gene RNA 
(Transcriptomics/Metabolomics), full blood on FTA dry-blood cards (Epigenomics) 
5 ml of EDTA blood is kept on ice and centrifuged within 60 min,10 min at 5000G and aliquoted. 
Snap-frozen and stored on dry ICE and then transfered to –80 °Celscius (°C) 
 
2,5 ml PAX gene RNA blood is stored in refrigerator for up to 5 days and then transferred to –80 °C 
FTA dry-blood cards are stored in individual plastic bags in room temperature or fridge +4-8°C.  
PExA Are kept on ICE, snap freezed and aliquoted, stored on dry ICE and then transfered to –80°C . 
Urine sampling: 5ml, kept on ICE, centrifuged 10min at 1000G and aliquoted. Snap-frozen and stored 
on dry ICE and then transfered to –80 °C.  
 
Venous blood samples that will be used to analyze fluid biomarkers of neuronal injury are collected in 
plasma EDTA (ethylenediaminetetraacetic acid) tubes and centrifuged for 15 minutes at 2400G and 4 
degrees centigrade. Directly after centrifugation, aliquots of 500μL serum will be frozen on dry ice 
and then stored at -78 degrees centigrade until analysed. 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
15 
 
 
 
Description of main analyses:  
Blood and Urine samples:  Transcriptomics to study changes in immune activation, stress responses, 
inflammation, and other biological processes at the mRNA level. Metabolomics to capture changes in 
energy metabolism, oxidative stress, inflammation, and nutrient processing. Proteinomics to quantify 
and characterize circulating proteins, including enzymes, cytokines, and signalling molecules, 
providing insights into systemic inflammation, stress responses, and regulatory pathways affected by 
the intervention. Epigenomics to assess methylation patterns, which may reflect immunological stress 
in response to the exposure. Lipidomics including phospholipids, sphingolipids, and fatty acids to 
uncover shifts in lipid metabolism, oxidative damage, and systemic stress responses. All –omics will 
used in conjunction with targeted, orthogonal, and quantitative methods to confirm/validate high-
throughput findings and explore novel biomarkers for inflammation and oxidative stress.  
PexA samples will be analyzed for lipid-profile using a a targeted LC-MSMS method, developed 
specifically for PexA. Proteins in PExA samples will be analysed with Olink (proteomics) but also a 
targeted protein-profile using mass-spectrometry. Fluid biomarkers of neuronal injury will be 
analyzed using either a NULISATM proteomics assay technology or a Neurology 4-Plex A assay on an 
HD-1 Single molecule array (Simoa) instrument. 
 
13.3 Self-reported symptoms  
 
Subjective assessment of shortness of breath (SOB) along with other respiratory symptoms will be 
assessed using a structured self-assessment form, specifically developed to detect early signs of 
pulmonary oxygen toxicity (POT). Participants will be asked to report whether they currently 
experience any of a predefined set of symptoms, including uncomfortable or labored breathing, 
cough, shortness of breath, increased respiratory effort, chest tightness or airway irritation. If a 
specific symptom is present, the severity of the symptom is graded on a numerical rating scale ranging 
from 0 (no discomfort) to 10 (worst imaginable discomfort). An open-ended category of “other 
symptom” is included to capture unexpected or atypical reactions. 
 
These assessments will be conducted among with PFTs and sampling biomarkers for oxidative stress, 
before and after each hyperbaric intervention to capture both immediate (acute) effects or any delayed 
onset of symptom as a response to the intervention. This custom designed scale provides a more 
symptom-specific assessment of objective pulmonary endpoints in our study protocol, replacing 
previously considered instruments such as the modified BORG-scale and VAS.
 
 
13. STATISTICAL ANALYSES 
Descriptive Statistics 
Baseline characteristics and outcome variables are summarized using descriptive statistics. 
Continuous variables will first be assessed for distribution, where normally distributed variables are 
presented as means with standard deviations (SD) and categorical variables will be summarized as 
frequencies and percentages. Rating scales, such as the BORG-scale and (VAS) for dyspnea, are 
considered ordinal variables and will therefore be reported as medians with interquartile ranges (IQR).  
Primary Outcome Analysis 
Difference in the absolute change in forced vital capacity (ΔFVC) between the two diving breathing 
gas exposures. ΔFVC is defined as the individual change from pre-exposure to post-exposure values 
within each condition. The main comparison will be the ΔFVC following hydrogen-enriched gas 
exposure versus control gas exposure within subjects. This will be analyzed using mixed effects 
regression models, Wilcoxon sign rank test or paired t-test. 
Secondary Outcome Analysis 
Additional lung function variables will be analyzed using mixed effects regression models, linear for 
continuous outcomes and logistic for categorical outcomes. The reversibility of FVC and FEV1 will 
be evaluated according to standard spirometric guidelines, assessing the following parameters:  

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
16 
 
 
• 
FVC (Forced Vital Capacity) 
• 
Slow Vital Capacity (SVC) 
• 
FEV1 (Forced Expiratory Volume in One Second) 
• 
FVC% (FVC as percentage of predicted value) 
• 
FEV1% (FEV1 as percentage of predicted value) 
• 
FEV1/FVC-ratio 
 
Results for fluid biomarkers of neuronal injury will be reported using mean (with standard deviation) 
and median (with both interquartile range and minimal/maximal) values. Both relative and absolute 
changes will be presented. Non-parametric statistical techniques will be used in analyses. 
Explorative analyses 
For the explorative analyses of lipid- and protein-profiles multivariate statistical methods will also be 
used including SIMCA and MUVR2 including Random Forest and Elastic Net.  
Statistical Significance 
A two-tailed p-value < 0.05 will be considered statistically significant. 
 
Sample Size Calculation 
To ensure a sufficient sample size, 32 participants are recruited for the study to account for potential 
dropouts or missing data. A minimum of 20 participants is required to achieve 80% statistical power 
with valid primary outcome data. 
Sample Size requirements 
The sample size estimation is based on a healthy 30-year-old male (180 cm, 75 kg) with an expected 
FVC of 7.2 L. Spirometry data, including (FVC) are calculated with a percentage of predicted values 
using Global Lung Initiative (GLI) reference standards. (27) 
• 
Relevant difference (Δ): 144 mL (2% change in FVC) 
• 
Expected standard deviation (SD) (σ): 216 mL 
• 
Significance level (α): 0.05 
• 
Power (1-β): 80%  
 
 
 
 
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
17 
 
 
15. ASSESMENT AND OUTCOME: 
To evaluate the effect of the intervention, a series of standardized measurements will be conducted at 
predefined time points in relation to each exposure (seen in Table 2). These assessments include both 
subjective and objective evaluations of pulmonary function, inflammatory markers, and potential 
adverse effects. Assessment of primary and secondary outcomes is designed to evaluate changes in 
oxidative stress, with within-participant comparisons made before and after the intervention. 
 
 
Assessment 
 
Description 
 
Pre 1st 
visit  
 
Post 
1st dive 
 
Post  
1st dive 
 
Pre 2nd 
dive  
 
Post  
2nd dive 
 
Post 2nd 
dive  
 
 
 
 
 
 
 
 
Date and time 
Tracking record of 
exposure timeline 
Before
1st dive  
30-120 
min 
12-36 h 
Before 
2nd dive  
30-120 
min  
12-36 h 
Anthropometric 
measurements 
Weight, height, sex, age, 
BMI 
X 
 
 
 
 
 
Self-reported 
symptoms 
SOB: Dyspnea etc.  
X 
X 
X 
X 
X 
X 
Oscillometry 
(Tremoflo) 
Airway resistance 
X 
X 
X 
X 
X 
X 
FeNO  
(Levels of NO) 
Airway inflammation 
X 
X 
X 
X 
X 
X 
Spirometry and body 
plethysmography 
FVC, FEV1, IC, FEF 25-
75%, PEF, TLC, RV, 
FRC 
X 
X 
X 
X 
X 
X 
DLCO 
Diffusion capacity 
X 
X 
X 
X 
X 
X 
PExA 
Airway lining fluid 
particle analysis 
X 
 
X 
X 
 
X 
Blood sample 
+ urin sample 
Analysis of oxidative 
stress, inflammatory 
biomarkers, biomarkers of 
neuronal injury 
X 
X 
X 
X 
X 
X 
Alveolar integrity  
Epithelial permeability 
(via DTPA uptake) 
 
 
X 
 
 
X 
Adverse events 
Systematic monitoring for 
safety 
X 
X 
X 
X 
X 
X 
 
 
Table 2. Time relationship for each measurement, in chronological order relative to each exposure. 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
18 
 
 
Spirometry and Body plethysmography (Δ FVC, SVC, FEV1, IC, FEF25-FEF50, FEF75, PEF, 
TLC, RV, FRC) 
As the spirometry results include assessment of forced vital capacity (FVC) as our primary outcome, 
these measurements play a central role in evaluating the intervention’s impact on pulmonary function. 
To provide a more comprehensive evaluation, additional spirometry parameters are included in our 
assessment; forced expiratory volume in one second (FEV1), forced expiratory flow, inspiratory 
capacity (IC) and peak expiratory flow (PEF). Pre- and post-exposure values are compared to 
determine whether hydrogen supplementation offers a protective effect on lung function. To further 
investigate the effects of hydrogen supplementation, we will examine its impact on: 
Diffusing Capacity for Carbon Monoxide (Δ DLCO) 
Measuring DLCO to assess the alveolar-capillary membrane and the lung’s gas exchange efficiency. A 
stable or improved DLCO post-dive in the H₂-supplemented group suggest a protective effect against 
the oxidative stress induced by high oxygen partial pressures. 
Exhaled Breath Particle Analysis (Δ PExA) 
PExA measurements will provide direct insight into biochemical changes within the epithelial lining 
fluid (ELF) of the lungs, potentially revealing early signs of pulmonary oxygen toxicity and the 
mitigating effects of hydrogen supplementation. 
Oscillometry (Tremoflo) 
Oscillometry will be performed to assess airway resistance and reactance, providing insight into 
potential changes in airway mechanics following exposure. 
Fractional Exhaled Nitric Oxide (FeNO) 
FeNO will be measured as a marker of airway inflammation, with pre- and post-exposure values 
analyzed to determine whether the intervention mitigates inflammatory responses associated with 
oxygen exposure. 
Self-Reported Symptoms (Dyspnea and SOB) 
Self-reported symptoms of dyspnea and shortness of breath (SOB) will be collected to capture 
subjective respiratory changes, allowing for correlation with objective pulmonary function measures. 
Biomarkers and blood samples:  
Blood samples will be analyzed to assess biomarkers of oxidative stress and systemic inflammation, 
enabling a comparative evaluation of metabolic responses before and after exposure to hyperbaric 
oxygen with or without hydrogen supplementation. Fluid biomarkers of neuronal injury will be 
analyzed to evaluate the effect of breathing hyperbaric oxygen, with and without addition of 
hydrogen, on the central nervous system.  
 
Adverse Events (AE) 
Adverse events (AE) will be systematically recorded throughout the study to ensure participant safety 
and assess any potential intervention-related effects, contributing to the overall risk-benefit evaluation 
of hydrogen supplementation. 
Serious Adverse Events (SAE) 
Given that the study follows a well-established safety protocol and remains within recognized 
exposure limits, the risk of serious adverse events (SAE) is considered low. However, any serious 
injuries or indications of an SAE will be systematically documented to ensure participant safety and 
evaluate potential risks associated with the intervention. In the event of an SAE, the participant will 
be advised to terminate their participation to mitigate further risk.
 
 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
19 
 
 
15. ETHICAL CONSIDERATIONS   
This study will be conducted in line with the International guidelines for good clinical practice (ICH-
GCP E6 (R2)(28) and the latest version of the Declaration of Helsinki.(29) All dive candidates will be 
clearly informed of their right to withdraw from the study at any time without any penalty. To ensure 
support and address any concerns, a designated researcher will be present during all study 
investigations. 
Ethical Review Authority 
The study has been reviewed and approved by the Swedish Ethical Review Authority 
(Etikprövningsmyndigheten) on September 10th, 2025 with the diary number: 2025-05124-01. 
Responsible applicant is the Swedish Armed Forces through the Diving and Naval Medicine Centre  
 
Registration and Reporting 
In addition to obtaining ethical approval, our study will prospectively be registered in a recognized 
clinical trial database (ClinicalTrials.gov) before recruitment of the first participant. Initially designed 
in accordance with Consolidated Standards of Reporting Trials (CONSORT) guidelines from 2010  
(30) and later on updated to the most recent CONSORT extensions and best practice standards as of 
2025 (31) to ensure transparency and publication of reliable results. 
Biological Materials for Future Research 
To ensure that all collected biological materials are handled in compliance with ethical and legal 
standard concerning participant privacy and data integrity, a biobank agreement will be established in 
accordance with the procedures of the Swedish Ethical Review Authority. The application will follow 
the Biobanks Act (2023:38)(32) and could only be submitted after EPM approval has been granted.  
Confidentiality and Data Protection 
All participant data will be handled with strict confidentiality. Data will be securely stored and 
accessible only to authorized study personnel. Personal identifiers will be removed, and data will be 
coded to ensure participant anonymity in compliance with applicable data protection regulations.  
 
16.1 Potential Risks: 
- Hyperbaric Exposure:  
Exposure to elevated pressures environments carries risks such as barotrauma to the ear or lungs. 
However, there is no risk of DCS due to the very low partial pressures of inert gases used. 
- Oxygen Toxicity:  
Neurological effects are unlikely at the partial pressures of oxygen planned for this experiment. 
Oxidative pulmonary stress effects are expected, but prior studies and experience have not 
demonstrated any long-term consequences following single exposures. (33) See also Section 12: Risk 
management.  
 Hydrogen mixed with oxygen poses a risk of explosion. See also Section 12: Risk management. 
- Handling of Personal Data:  
Ensuring secure and ethical management of sensitive participant data. 
16.2 Risk Mitigation Measures: 
- Hyperbaric Exposures:  
The experiments will adhere to established military safety protocols. Additionally, the participants are 
experienced military divers, which minimizes the risk of barotrauma. We will remain within national 
oxygen exposure limits for military diving. Furthermore, participants are military divers Assessed by 
a physician to have healthy lungs. 
- Hydrogen and Oxygen Mixtures:  
It’s well known that hydrogen can be explosive when mixed with oxygen, thus, this reaction doesn’t 
occur if the hydrogen fraction is kept sufficiently low. Gas cylinders will be pre-filled according to 
current standards to ensure an adequately low hydrogen fraction. See also Section 12: Risk 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
20 
 
 
management. 
Handling of Personal Data:  
Data will be pseudo-anonymized, and individual keys will be securely stored in a safe at Blekinge 
Institute of Technology.
17. Potential Benefits: 
- Potential Intervention Development: 
The findings could contribute to reducing the toxic effects of oxygen on tissues, enabling dives that 
are impossible today because of high inert gas load and/or to high pressure of oxygen. Additionally, 
such interventions could improve treatments for critically ill patients (e.g., intensive care unit)  
- Improved Understanding: 
The experiment may provide a better understanding of the toxic effects and pathophysiological effects 
of oxygen on lung tissues. We assess that the potential benefits outweigh the risks in this study setup. 
The planned exposures are no greater than those already approved, and our intervention and outcomes 
are likely to provide valuable knowledge for both divers and patients. 
- Enabling Longer and Safer Dives: 
By mitigating oxygen toxicity, hydrogen-enriched gas mixtures may enable longer and deeper dives 
than currently possible. Reducing the oxygen toxicity risk could allow divers to safely increase the 
exposure time at high pressures, optimizing operational efficiency and reducing decompression-
related constraints. A higher possible oxygen fraction also means a lower inert gas fraction, further 
decreasing nitrogen absorption and reducing the risk of decompression sickness 
- Improved Clinical Applications 
The ability to reduce oxygen toxicity could have profound medical implications, particularly in 
neonatal care, intensive care and post-organ transplantation. Basically, all areas where oxygen therapy 
is crucial but carries risks of oxidative damage. Understanding how to modulate oxidative stress in a 
controlled setting could enhance critical care treatments, potentially improving patient outcomes in 
high-oxygen environments. 
 
Risk-Benefit Assessment 
We assess that the potential benefits outweigh the risks in this study setup. The planned oxygen 
exposure strictly adheres to diving regulations set by the Swedish Armed Forces’ and does not exceed 
levels approved in existing hyperbaric research and clinical practice. These standards are specifically 
designed to ensure participant safety during hyperbaric regulations. Furthermore, hydrogen has been 
repeatedly incorporated into breathing mixtures in professional deep-sea diving to safely reduce the 
concentration of oxygen or other active components.  
 
The practice of using hydrogen as a diluent gas is well-documented in several experimental programs 
who demonstrated its feasibility under hyperbaric conditions simulated at depths of up to 700 meters 
(19). As outlined above, the planned oxygen exposure of 1.7 ATA in our study is well below the 
limits investigated in previous hyperbaric research. It can therefore be concluded that the exposure 
falls within established safety margins in accordance with existing scientific evidence and diving 
regulations. Given the high potential for both scientific and clinical impact, the results of our study are 
expected to provide valuable insights for diving professionals as well as for the care of critically ill 
patients requiring high oxygen concentrations. 
 
18. Summary  
The antioxidative potential of hydrogen gas presents a promising strategy for reducing pulmonary and 
neurological oxygen toxicity, reaching implications for diving safety. This study aims to explore the 
feasibility of the antioxidative potential of hydrogen, proposing a novel approach to safer diving 
protocols and improved clinical interventions. Specifically, it aims to assess whether incorporation of 
a small fraction of hydrogen into a breathing gas could effectively reduce symptoms of POT within 
oxygen-enriched diving exposed to elevated partial pressure of 1.75 ATA over 240 minutes. If 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
21 
 
 
successful, this approach could provide several key benefits, including extended dive durations by 
mitigating the limitations of oxygen toxicity. 
WORK PLAN:  
1) Study preparation: 2025 
2) Recruitment and data collection: 2026-2027.  
3) Analysis and reporting: 2027-2030
19. GUIDELINES  
The study will be conducted under the oversight of the Swedish Armed Forces Diving and Naval 
Medicine Centre (DNC), ensuring full compliance with internationally recognized hyperbaric safety 
standards. The Diving Medicine Section is responsible for all medical aspects of diving within the 
Swedish Armed Forces, including research and operational support. By adhering to guidelines set by 
the European Diving Technology Committee (EDTC) and the European Committee for Hyperbaric 
Medicine (ECHM) upholding highest standards of protection and operational integrity. (34) To ensure 
both safety and reliability within our study while meeting the highest medical and scientific standards. 
 
20. DISSEMINATION 
Findings will be published in peer-reviewed journals and presented at international diving and 
medical conferences. Planned publications include of: 1) primary outcome and main secondary 
outcomes; 2) PeXA outcomes; 3) blood inflammatory profile outcomes; 4) exploratory analyses of 
mechanisms underlying oxygen toxicity. Authorship will be determined in accordance with guidelines 
from the International Committee of Medical Journal Editors (www.icmje.org/). Further, relevant 
professionals and organizations in diving medicine will be informed through targeted 
communications, to ensure the results contribute to both scientific advancements and practical 
applications in the field.
22. BUDGET AND RESOURCES 
Current Funding: The project is supported by grant 5005113/22FMV2951. 
Key Resources and Collaborations: 
Hyperbaric Facilities:  
Access to hyperbaric chambers and associated medical support is secured through a collaboration with 
DNC, Karlskrona. Including the availability of a certified physician during procedures. 
 
Measurement Equipment:  
Required instrumentation and technical equipment will be provided through collaboration with the 
Blekinge Institute of Technology, Department of Translational Medicine, Lund University and 
Occupational and Environmental Medicine, Sahlgrenska Academy 
 
Personnel: The research team includes physicians with expertise in diving medicine, pulmonary 
physiology, nuclear medicine and environmental medicine, combined with specialists in advanced 
marine systems engineering. The team further comprises research assistants and biostatisticians with 
additional technical and administrative personnel as needed throughout the project. 
 
REFERENCES:  
 
1. RMS-Dyk : regler för militär sjöfart. Stockholm: Försvarsmakten; 2013.  
2. Preece R. Pulmonary oxygen toxicity in occupational diving. Occup Med Oxf Engl. 2023 Dec 
29;73(8):518.  

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
22 
 
 
3. Ahti PA, Wikgren J. Rapture of the deep: gas narcosis may impair decision-making in scuba 
divers. Diving Hyperb Med. 2023 Dec 20;53(4):306–12.  
4. Bao XC, Yang T, Fang YQ, Sun YJ, Wang N. Lung function changes in divers after a single deep 
helium-oxygen dive. Diving Hyperb Med. 2022 Sep 30;52(3):183–90.  
5. Šegrt Ribičić I, Valić M, Božić J, Obad A, Glavaš D, Glavičić I, et al. Influence of oxygen 
enriched gases during decompression on bubble formation and endothelial function in self-
contained underwater breathing apparatus diving: a randomized controlled study. Croat Med J. 
2019 Jun 13;60(3):265–72.  
6. Wilmshurst P, Bryson P. Relationship between the clinical features of neurological decompression 
illness and its causes. Clin Sci Lond Engl 1979. 2000 Jul;99(1):65–75.  
7. de Jong FJM, Wingelaar TT, van Hulst RA. Pulmonary oxygen toxicity in occupational diving. 
Occup Med Oxf Engl. 2023 Jun 26;73(5):231–2.  
8. Fothergill DM, Gertner JW. Exhaled Nitric Oxide and Pulmonary Oxygen Toxicity Susceptibility. 
Metabolites. 2023 Aug 8;13(8):930.  
9. de Jong FJM, Wingelaar TT, van Hulst RA. Pulmonary oxygen toxicity in occupational diving. 
Occup Med Oxf Engl. 2023 Jun 26;73(5):231–2.  
10. 
Klitgaard TL, Schjørring OL, Nielsen FM, Meyhoff CS, Perner A, Wetterslev J, et al. Higher 
versus lower fractions of inspired oxygen or targets of arterial oxygenation for adults admitted to 
the intensive care unit. Cochrane Database Syst Rev. 2023 Sep 13;9(9):CD012631.  
11. 
Risberg J, van Ooij PJ. Hyperoxic exposure monitoring in diving: A farewell to the UPTD. 
Undersea Hyperb Med J Undersea Hyperb Med Soc Inc. 2022;49(4):395–413.  
12. 
van Ooij PJ a. M, van Hulst RA, Houtkooper A, Sterk PJ. Nitric oxide and carbon monoxide 
diffusing capacity after a 1-h oxygen dive to 9 m of sea water. Clin Physiol Funct Imaging. 2014 
May;34(3):199–208.  
13. 
Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, et al. 
Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat 
Med. 2007 Jun;13(6):688–94.  
14. 
Yu J, Yu Q, Liu Y, Zhang R, Xue L. Hydrogen gas alleviates oxygen toxicity by reducing 
hydroxyl radical levels in PC12 cells. PloS One. 2017;12(3):e0173645.  
15. 
Kawamura T, Wakabayashi N, Shigemura N, Huang CS, Masutani K, Tanaka Y, et al. 
Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am J Physiol Lung Cell 
Mol Physiol. 2013 May 15;304(10):L646-656.  
16. 
Cole AR, Perry DA, Raza A, Nedder AP, Pollack E, Regan WL, et al. Perioperatively Inhaled 
Hydrogen Gas Diminishes Neurologic Injury Following Experimental Circulatory Arrest in Swine. 
JACC Basic Transl Sci. 2019 Apr;4(2):176–87.  
17. 
Harris RJ, Challen CJ, Mitchell SJ. The first deep rebreather dive using hydrogen: case report. 
Diving Hyperb Med. 2024 Mar 31;54(1):69–72.  
18. 
Sun Q, Han W, Nakao A. Selective Antioxidative Effect of Hydrogen. In: Sun X, Ohta S, 
Nakao A, editors. Hydrogen Molecular Biology and Medicine [Internet]. Dordrecht: Springer 
Commented [AL1]: Samma referens som nr. 7? 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
23 
 
 
Netherlands; 2015 [cited 2025 Feb 13]. p. 61–80. Available from: https://doi.org/10.1007/978-94-
017-9691-0_5 
19. 
Lafay V, Barthelemy P, Comet B, Frances Y, Jammes Y. ECG changes during the 
experimental human dive HYDRA 10 (71 atm/7,200 kPa). Undersea Hyperb Med J Undersea 
Hyperb Med Soc Inc. 1995 Mar;22(1):51–60.  
20. 
Dive Manual Rev 7 Change A.pdf [Internet]. [cited 2025 Apr 11]. Available from: 
https://www.navsea.navy.mil/Portals/103/Documents/SUPSALV/Diving/Dive%20Manual%20Rev
%207%20Change%20A.pdf 
21. 
Yıldız F, LeBaron TW, Alwazeer D. A comprehensive review of molecular hydrogen as a 
novel nutrition therapy in relieving oxidative stress and diseases: Mechanisms and perspectives. 
Biochem Biophys Rep. 2025 Mar;41:101933.  
22. 
Wellek S, Blettner M. On the Proper Use of the Crossover Design in Clinical Trials. Dtsch 
Ärztebl Int. 2012 Apr;109(15):276–81.  
23. 
Model 6800: User instructions. https://vitalograph.com/storage/uploads/74472a0a-bd4c-4261-
acb6-38c690939ad3/09000_3_PneumotracMKIV_IFU_ENG_SCREEN.pdf [Internet]. [cited 2025 
May 6]. Available from: https://vitalograph.com/storage/uploads/74472a0a-bd4c-4261-acb6-
38c690939ad3/09000_3_PneumotracMKIV_IFU_ENG_SCREEN.pdf 
24. 
Graham BL, Steenbruggen I, Miller MR, Barjaktarevic IZ, Cooper BG, Hall GL, et al. 
Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European 
Respiratory Society Technical Statement. Am J Respir Crit Care Med. 2019 Oct 15;200(8):e70–88.  
25. 
Graham BL, Steenbruggen I, Miller MR, Barjaktarevic IZ, Cooper BG, Hall GL, et al. 
Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European 
Respiratory Society Technical Statement. Am J Respir Crit Care Med. 2019 Oct 15;200(8):e70–88.  
26. 
King GG, Bates J, Berger KI, Calverley P, Melo PL de, Dellacà RL, et al. Technical standards 
for respiratory oscillometry. Eur Respir J [Internet]. 2020 Feb 27 [cited 2025 Jun 3];55(2). 
Available from: https://publications.ersnet.org/content/erj/55/2/1900753 
27. 
Cooper BG, Stocks J, Hall GL, Culver B, Steenbruggen I, Carter KW, et al. The Global Lung 
Function Initiative (GLI) Network: bringing the world’s respiratory reference values together. 
Breathe Sheff Engl. 2017 Sep;13(3):e56–64.  
28. 
Agency EM. ICH E6 Good clinical practice - Scientific guideline | European Medicines 
Agency (EMA) [Internet]. 2025 [cited 2025 Mar 20]. Available from: 
https://www.ema.europa.eu/en/ich-e6-good-clinical-practice-scientific-guideline 
29. 
Goodyear MDE, Krleza-Jeric K, Lemmens T. The Declaration of Helsinki. BMJ. 2007 Sep 
29;335(7621):624–5.  
30. 
Dwan K, Li T, Altman DG, Elbourne D. CONSORT 2010 statement: extension to randomised 
crossover trials. 2019 Jul 31 [cited 2025 Feb 25]; Available from: 
https://www.bmj.com/content/366/bmj.l4378 
31. 
Hopewell S, Chan AW, Collins GS, Hróbjartsson A, Moher D, Schulz KF, et al. CONSORT 
2025 statement: updated guideline for reporting randomised trials. The Lancet. 2025 May 
3;405(10489):1633–40.  
Commented [AL2]: Samma referens som nr. 24? 

Research Plan v. 2.1 [2025-10-27]: Effect of Hydrogen Gas on Hyperbaric Oxygen Toxicity 
 
24 
 
 
32. 
Biobankslag (2023:38) [Internet]. [cited 2025 Feb 25]. Available from: 
https://www.riksdagen.se/sv/dokument-och-lagar/dokument/svensk-
forfattningssamling/biobankslag-202338_sfs-2023-38/ 
33. 
Abraini JH, David HN, Vallée N, Risso JJ. Theoretical considerations on the ultimate depth 
that could be reached by saturation human divers. Med Gas Res. 2016;6(2):119–21.  
34. 
Mathieu D, Marroni A, Kot J. Tenth European Consensus Conference on Hyperbaric 
Medicine: recommendations for accepted and non-accepted clinical indications and practice of 
hyperbaric oxygen treatment. Diving Hyperb Med. 2017 Mar;47(1):24–32.