Molecular Hydrogen in Redox Biology and Human Health: Mechanistic Insights, Clinical Evidence, and Translational Prospects for Sublingual Delivery

1. Introduction

Molecular hydrogen (H₂) was first recognized as a biologically active antioxidant following the landmark discovery that H₂ selectively scavenges highly cytotoxic reactive oxygen species (ROS), particularly hydroxyl radicals (•OH) and peroxynitrite, without disrupting physiological redox signaling [1]. The recognition of molecular hydrogen (H₂) as a biologically active molecule with antioxidant properties has led to the development of a research area commonly referred to as hydrogen medicine [1,2]. A pivotal study by Ohsawa et al., (2007) demonstrated that H₂ selectively reduces highly cytotoxic hydroxyl radicals while exerting minimal effects on other reactive oxygen species (ROS) involved in physiological signaling, thereby conferring protection against ischemia-reperfusion injury in experimental models. This foundational study demonstrated that H₂ penetrates membranes rapidly and mitigates oxidative injury in cerebral ischemia, establishing H₂ as a selective antioxidant gas rather than a conventional broad-spectrum radical scavenger [1]. Since this initial observation, increasing attention has been directed toward the potential role of molecular hydrogen in conditions associated with oxidative and inflammatory stress [3,4]. Subsequent work clarified that hydrogen does not merely neutralize ROS but modulates redox-sensitive signaling pathways, influencing gene expression, mitochondrial resilience, and inflammatory cascades [5].

In contrast to conventional antioxidants, which typically act through broad and non-selective scavenging of ROS, molecular hydrogen possesses unique physicochemical characteristics, including its small molecular size, electrical neutrality, and high diffusibility [2]. These properties enable H₂ to rapidly penetrate cellular membranes and subcellular compartments and, based on preclinical evidence, to cross the blood–brain barrier [6]. Such features have prompted interest in H₂ as a redox modulator rather than a classical antioxidant, with the theoretical advantage of mitigating oxidative damage while preserving essential redox signaling pathways [1,7].

Human studies investigating molecular hydrogen have largely focused on administration via hydrogen-rich water, inhalation of hydrogen gas, or hydrogen-enriched solutions used in clinical settings such as hemodialysis [2,4,8]. Across these modalities, changes have been reported in biomarkers related to oxidative stress, inflammation, cardiometabolic risk, and immune function [9,10,11]. However, these findings are characterized by substantial heterogeneity in study design, dosing regimens, exposure duration, and target populations, resulting in variable effect sizes and limited reproducibility [3,12,13]. Accordingly, although available evidence supports biological activity of H₂ in humans, definitive conclusions regarding clinical efficacy remain limited.

To date, sublingual delivery of molecular hydrogen has not been examined in peer-reviewed clinical studies. Nonetheless, the sublingual route is widely utilized for small, rapidly diffusible molecules due to its dense vascularization and potential to bypass gastrointestinal metabolism [2]. This review synthesizes current mechanistic and clinical evidence related to molecular hydrogen with a focus on redox biology and examines the physicochemical rationale and translational considerations for exploring sublingual administration as a potential alternative delivery route. Emphasis is placed on distinguishing established evidence from hypothesis-driven concepts and on identifying key gaps that must be addressed through rigorous experimental and clinical investigation.

2. Mechanistic Insights: Established and Emerging Concepts

2.1. Selective Redox Interactions

Molecular hydrogen (H₂) is increasingly recognized as a selective redox-active molecule that preferentially targets highly cytotoxic reactive species while largely preserving physiologic redox signaling. Chemically, this selectivity refers to the ability of H₂ to efficiently react with hydroxyl radicals (•OH) and, to a lesser extent, peroxynitrite (ONOO⁻)-derived species, while showing minimal reactivity toward superoxide (O₂•⁻), hydrogen peroxide (H₂O₂), and nitric oxide (NO•) under physiological conditions [1,14,15,16]. Quantum chemical and energetic analyses support this phenomenon by demonstrating that the reaction between H₂ and •OH has a sufficiently low activation energy to occur in vivo, whereas reactions with O₂•⁻ and H₂O₂ possess much higher energy barriers, rendering them kinetically unfavorable [17]. Monte Carlo track-chemistry simulations in irradiated water further confirm that dissolved H₂ selectively scavenges •OH radicals. Although H₂ is less potent than conventional radioprotective agents such as cystamine, it demonstrates a substantially superior safety profile [18].

In addition to hydroxyl radicals, H₂ also modulates reactive nitrogen species, particularly peroxynitrite-related oxidants. Experimental studies demonstrate that H₂ reduces protein tyrosine nitration and attenuates cell death in chondrocytes and retinal tissues exposed to nitric oxide donors, while simultaneously restoring disease-relevant gene expression profiles [18,19]. Importantly, H₂ appears to neutralize harmful nitrosative species without interfering with physiological nitric oxide signaling, thereby preserving essential vascular and cellular functions [20,21].

Beyond direct chemical scavenging, H₂ exerts broader regulatory effects on intracellular redox signaling pathways. Numerous studies show that H₂ activates redox-sensitive cytoprotective pathways, particularly the Nrf2/Keap1 axis, leading to upregulation of endogenous antioxidant enzymes and modulation of inflammatory and apoptotic responses [16,20,21]. One proposed mechanistic model identifies oxidized Fe-porphyrins, such as hematin, as intermediary redox targets. In this model, •OH initially oxidizes heme structures, after which H₂ reacts with hematin-OH complexes, facilitating catalytic conversion of •OH into H₂O and promoting downstream activation of Nrf2 signaling through oxidative modification of Keap1 [22]. Collectively, these findings support the concept that H₂ functions not merely as a simple radical scavenger, but as a selective redox modulator capable of preserving physiologic signaling while mitigating oxidative and nitrosative damage.

The proposed mechanisms by which molecular hydrogen influences redox biology including selective radical interactions, modulation of redox-sensitive signaling pathways, and downstream anti-inflammatory effects are summarized in Figure 1.

2.2. Anti-Inflammatory and Anti-Apoptotic Effects

Molecular hydrogen (H₂) has consistently demonstrated anti-inflammatory and anti-apoptotic properties across a wide range of experimental disease models. The anti-inflammatory actions of H₂ are strongly associated with suppression of pro-inflammatory cytokines and signaling pathways. Experimental studies show that H₂ decreases the production of inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, and high-mobility group box 1 (HMGB1), while enhancing anti-inflammatory cytokines including IL-10. These effects have been observed in models of sepsis [23,24], intestinal inflammation [25]23,24,[25]] neurodegeneration [26,27], ultraviolet B (UVB)-induced skin injury [28], and COVID-19-associated inflammation [29]. Mechanistically, H₂ suppresses activation of nuclear factor-kappa B (NF-κB) and inhibits phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Through these actions, H₂ reduces transcription of inflammatory genes in macrophages, endothelial cells, intestinal tissues, neural tissues, and skin [27,29].

In parallel, H₂ activates endogenous antioxidant defense systems, particularly the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway. Activation of Nrf2 leads to upregulation of phase II antioxidant and cytoprotective enzymes, including HO-1, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Through modulation of the Nrf2/HO-1 axis, H₂ protects lung endothelial cells and neural tissues from septic and hypoxic-ischemic injury while simultaneously attenuating oxidative stress and inflammatory signaling [26,27,29].

Furthermore, H₂ exerts significant anti-apoptotic effects, particularly through preservation of mitochondrial integrity and inhibition of mitochondria-dependent cell death pathways. In myocardial infarction models, inhaled H₂ preserved mitochondrial membrane potential, increased ATP production, restored mitochondrial complex I and III activities, and reduced oxidative damage markers such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA). These changes were accompanied by normalization of cytochrome c release, decreased Bax/Bcl-2 ratio, and reduced activation of cleaved caspase-9 and caspase-3, collectively indicating inhibition of intrinsic apoptotic signaling [30].

Protective anti-apoptotic effects have also been observed in neurologic injury models. In neonatal hypoxic-ischemic encephalopathy, H₂ reduced infarct volume, cerebral edema, apoptosis, and neurobehavioral deficits in Nrf2-competent mice. These effects were associated with decreased ROS and MDA levels, reduced inflammatory cytokines, and suppression of NF-κB and NLRP3 inflammasome activation [26]. In neurodegenerative disease models such as 5xFAD mice, H₂ lowered the BAX/BCL-2 ratio and preserved neuronal markers alongside its anti-inflammatory and antioxidant actions [27]. Broader reviews similarly identify H₂ as anti-apoptotic in cardiovascular, radiation-induced, and ischemia-reperfusion injuries, although the precise molecular targets remain incompletely characterized [5,29,30].

Emerging clinical and translational studies provide preliminary support for these mechanisms. In sepsis and COVID-19 patients, inhaled H₂ or H₂/O₂ gas mixtures have been associated with improved survival, reduced respiratory symptoms, and improved clinical status, findings consistent with anti-inflammatory and anti-apoptotic effects [29,30]. Experimental cardiovascular and dermatologic studies likewise support translational potential [28,29,30]. Nevertheless, current human studies remain relatively small, and large-scale controlled clinical trials incorporating apoptosis-specific and mechanistic biomarkers are still needed to establish definitive therapeutic efficacy and molecular targets of H₂ in human disease.

2.3. Mitochondrial Protection and Redox Homeostasis

Molecular hydrogen (H₂) has emerged as an important regulator of mitochondrial function and cellular redox homeostasis through both direct and indirect mechanisms. Experimental evidence from in vitro systems, animal studies, and integrative reviews indicates that H₂ influences mitochondrial bioenergetics, oxidative stress responses, and redox-sensitive signaling pathways. Rather than functioning solely as a conventional antioxidant, H₂ is increasingly viewed as a modulator of mitochondrial signaling and adaptive redox regulation.

One of the most consistently reported effects of H₂ is the enhancement of mitochondrial bioenergetic function. In SH-SY5Y neuroblastoma cells, H₂ increases mitochondrial membrane potential (Δψm), intracellular ATP levels, and state-3 oxygen consumption without altering mitochondrial DNA copy number, suggesting improved efficiency of oxidative phosphorylation (OXPHOS) rather than increased mitochondrial biogenesis. H₂ also demonstrates protective effects under oxidative stress conditions. Pretreatment with H₂, but not post-treatment, protects SH-SY5Y cells against H₂O₂-induced cell death, indicating that adaptive cellular responses may be required for protection [31]. Similarly, in melanocytes exposed to oxidative stress, H₂ preserves mitochondrial morphology, maintains Δψm and ATP production, and restores cytochrome b and cytochrome c expression while reducing apoptosis [32].

Protective mitochondrial effects of H₂ have also been observed following ischemic and hypoxic injury. In intestinal Caco-2 cells subjected to hypoxia-reoxygenation, hydrogen gas restores mitochondrial membrane potential, ATP production, and oxygen consumption while reducing reactive oxygen species (ROS) generation and pro-apoptotic signaling [33]. In models of subarachnoid hemorrhage and chronic stress, H₂ promotes mitochondrial quality control through activation of the NRF2-PINK1/Parkin pathway, enhancing mitophagy and preserving mitochondrial homeostasis [34,35]. These findings suggest that H₂ supports the removal of damaged mitochondria and maintenance of a healthy mitochondrial network during cellular stress.

A central feature of H₂ biology is its role in maintaining redox homeostasis through activation of endogenous antioxidant systems. Multiple studies demonstrate that H₂ activates the Keap1-Nrf2-antioxidant response element (ARE) signaling pathway, leading to increased expression of antioxidant and phase II detoxification enzymes such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and NAD(P)H quinone oxidoreductase 1 (NQO1) [15,31,36,37]. These effects improve the cellular glutathione redox balance, increase the GSH/GSSG ratio, and reduce lipid peroxidation [31].

Emerging evidence further supports the concept of H₂ as a mitohormetic or “redox adaptogen” molecule. In SH-SY5Y cells, H₂ exposure mildly increases mitochondrial superoxide production and transiently lowers the GSH/GSSG ratio, changes that subsequently activate Nrf2 signaling and stimulate adaptive antioxidant defenses [31]. Importantly, the cytoprotective effects require pretreatment time, supporting the idea that H₂ induces an adaptive stress response rather than acting solely as an immediate antioxidant. Consequently, several reviews now describe H₂ as a “redox homeostasis regulator” or “mitochondria-targeting nutrient” rather than a simple free radical scavenger [15,36,37].

The above findings support a shift in perspective regarding the biological actions of H₂. While early research emphasized direct scavenging of hydroxyl radicals, accumulating evidence suggests that direct radical neutralization alone is insufficient to explain the broad protective effects observed experimentally. Instead, mitochondrial redox proteins, adaptive antioxidant signaling pathways, and modulation of mitochondrial electron transport appear to represent primary mechanisms through which H₂ preserves mitochondrial integrity, regulates redox homeostasis, and protects cells from oxidative and inflammatory injury.

2.4. Endothelial Function and Inflammation Modulation

Molecular hydrogen (H₂) has been increasingly investigated in vascular and neurovascular disorders characterized by oxidative stress, inflammation, and endothelial dysfunction.

One of the major vascular effects of H₂ is the improvement of endothelial function through modulation of nitric oxide bioavailability and endothelial nitric oxide synthase (eNOS) activity. In models of obstructive sleep apnea–hypopnea syndrome (OSAHS), intermittent hypoxia induced significant endothelial dysfunction characterized by elevated reactive oxygen species (ROS), increased malondialdehyde (MDA) levels, impaired endothelium-dependent vasodilation, and reduced NO signaling. Treatment with H₂-rich medium in vitro or inhaled 2% H₂ gas in rats significantly reduced ROS and MDA accumulation, restored endothelial function, and improved vasodilation by approximately 40%. Mechanistically, H₂ promoted eNOS recoupling through increased phosphorylation of eNOS at Ser1177 and preservation of the tetrahydrobiopterin/dihydrobiopterin (BH₄/BH₂) ratio, thereby restoring NO bioavailability and improving vascular responsiveness [38].

In addition to functional improvements, H₂ also attenuated structural vascular remodeling induced by intermittent hypoxia. Experimental studies demonstrated reductions in vascular medial thickening and collagen deposition following H₂ treatment, suggesting that H₂ provides both functional and structural protection to the vascular endothelium under chronic oxidative stress conditions [38].

H₂ also exerts potent anti-inflammatory effects within endothelial tissues. In lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs) and polymicrobial sepsis mouse models, H₂-rich medium significantly reduced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and high-mobility group box 1 (HMGB1), while simultaneously increasing levels of the anti-inflammatory cytokine IL-10. These findings indicate that H₂ suppresses endothelial inflammatory activation and leukocyte adhesion processes that contribute to vascular injury and dysfunction [38].

Many of these anti-inflammatory effects appear to depend on activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway. Studies showed that H₂ upregulated HO-1 expression through Nrf2 activation, and inhibition of HO-1 abolished the suppressive effects of H₂ on adhesion molecules and inflammatory cytokines [38]. This suggests that the Nrf2/HO-1 axis is a critical mediator of H₂-induced endothelial protection. Similar anti-inflammatory effects were observed in OSAHS models, where H₂ inhalation reduced TNF-α and ICAM-1 expression in aortic tissue in parallel with improvements in endothelial function [38].

Current vascular studies support several interconnected mechanistic pathways through which H₂ exerts endothelial protection. These include reduction of oxidative stress markers such as ROS and MDA, restoration of eNOS activity and NO bioavailability, activation of Nrf2-dependent antioxidant signaling, suppression of inflammatory cytokines and adhesion molecules, and reduction of endothelial apoptosis. Through these mechanisms, H₂ improves endothelial viability and preserves vascular integrity in conditions associated with oxidative and inflammatory injury [15,38,39].

Broader reviews of H₂ in cardiovascular disease consistently emphasize its antioxidant, anti-inflammatory, and anti-apoptotic properties, particularly those mediated through Nrf2-centered redox signaling ([5,15]. However, important gaps remain. The direct molecular targets of H₂ within endothelial cells and the precise hierarchy of signaling pathways involved are not yet fully defined. Likewise, while contemporary reviews of endothelial dysfunction extensively discuss oxidative stress, NO depletion, eNOS uncoupling, NLRP3 inflammasome activation, and adhesion molecule signaling in vascular disease, H₂ has not yet been systematically incorporated into standard endothelial-protective therapeutic frameworks [40,41,42]

Current evidence suggests that H₂ exerts endothelial-protective effects primarily through restoration of nitric oxide (NO) signaling, reduction of oxidative injury, suppression of inflammatory activation, and preservation of endothelial cell viability. Although most data are derived from experimental and preclinical models, the findings collectively support a potentially important role of H₂ in vascular homeostasis and cardiovascular protection. Consequently, further mechanistic and clinical studies are needed to clarify the translational role of H₂ in vascular medicine and endothelial dysfunction-related disorders.

2.5. Physicochemical Properties and Formulation Rationale

Molecular hydrogen possesses distinctive physicochemical properties that underpin its proposed biological activity and influence its suitability for different delivery routes [2]. As the smallest diatomic molecule, H₂ is nonpolar, electrically neutral, and characterized by rapid diffusion through aqueous solutions, lipid membranes, and biological tissues [2,43]. In aqueous media, the solubility of hydrogen at standard atmospheric pressure and room temperature is approximately 1.6 mg/L (1.6 ppm) [44]. Hydrogen-rich water produced by electrolysis or other enrichment methods typically achieves dissolved hydrogen concentrations ranging from approximately 100 to 1,300 ppb immediately after generation, with concentrations declining over time due to rapid outgassing. Midrange concentrations (~600 ppb) are often considered feasible formulation targets, although optimal concentrations for specific biological effects have not been definitively established [45].

Hydrogen-enriched aqueous solutions frequently exhibit a negative oxidation–reduction potential (ORP), commonly ranging from −100 to −750 mV. Importantly, ORP reflects the presence of dissolved hydrogen rather than serving as a quantitative measure of antioxidant capacity or biological potency, and it should not be used as a surrogate for hydrogen concentration [46,47].

The physicochemical behavior of dissolved hydrogen has important implications for formulation stability, delivery efficiency, and measurement strategies. These considerations including rapid outgassing, limitations of ORP as a proxy measure, and the importance of point-of-use concentration, are summarized in Figure 2.

The sublingual route provides a highly vascularized mucosal surface that facilitates rapid absorption of small, diffusible molecules into the systemic circulation, potentially bypassing gastrointestinal transit and first-pass metabolism [48,49]. Although no pharmacokinetic data exist for sublingual hydrogen delivery, hydrogen’s small size and diffusibility suggest theoretical compatibility with this route. However, its volatility and rapid dissipation present unique formulation and delivery challenges [2].

Furthermore, as an example, the “Travel Water Spray” (TWS) developed by Travel Water Global Research Group (USA), may be conceptualized as a portable micro-spray platform designed to deliver dissolved molecular hydrogen via oral mucosal exposure. This approach represents both a behavioral and logistical innovation, enabling frequent, low-volume administration while avoiding the need for large fluid intake. Such a delivery model may support real-world adherence and aligns with emerging micro-dosing strategies aimed at maintaining redox homeostasis. Within this framework, TWS can be positioned as a potential daily-use tool to support mitochondrial redox balance, endothelial signaling, and the resolution of inflammatory processes. However, this concept remains a translational hypothesis and requires rigorous validation. Future studies should incorporate breath hydrogen pharmacokinetics, oxidative stress biomarkers, endothelial function panels, and pragmatic adherence trials to determine feasibility, bioavailability, and clinical relevance.

3. Clinical Evidence and Translational Relevance

Although sublingual delivery of molecular hydrogen has not been evaluated in peer-reviewed clinical studies, evidence from other administration routes provides context for its potential biological relevance [2,5]. Human investigations to date have primarily employed hydrogen-rich water (HRW), inhalation of hydrogen gas, or hydrogen-enriched solutions used in medical settings such as hemodialysis [2,8]. Across these modalities, outcomes have largely focused on biomarkers of oxidative stress, inflammation, metabolic regulation, and immune function [9,11].

Randomized controlled trials and controlled pilot studies of HRW have reported reductions in oxidative stress markers and inflammatory mediators in adults with metabolic risk factors, alongside changes in lipid profiles and glycemic indices [8,9]. For example, several trials have observed improvements in antioxidant capacity, reductions in low-density lipoprotein cholesterol, and modest changes in glycemic control following sustained HRW intake [5,8].

More recently, a double-blind randomized placebo-controlled study by Zhang et al., (2025) further demonstrated attenuation of oxidative stress and inflammation in patients with chronic high-altitude disease, supporting the biological activity of H₂ across diverse clinical populations [50]. However, not all studies report consistent results; a multicenter double-blind RCT by Ogawa et al., (2022) found no significant improvement in overall insulin resistance among patients with type 2 diabetes, though subgroup analysis suggested benefit in those with higher baseline insulin resistance [51]. The authors attributed these null findings in part to the small sample size and called for larger-scale, longer-term trials to verify the effects of electrolyzed hydrogen-rich water in T2DM patients, noting that current evidence remains insufficient to draw definitive conclusions [50].

Similarly, a randomized double-blind trial in healthy adults by Sim et al.,(2020) reported no significant between-group differences in serum oxidative stress markers, including derivatives of reactive oxygen and 8-OHdG, following four weeks of HRW consumption [11], suggesting that the magnitude of hydrogen’s redox effects may be influenced by baseline oxidative burden and disease status. Collectively, these findings reflect heterogeneity in study design, hydrogen concentration, intervention duration, and participant characteristics, and underscore the need for more standardized clinical investigation [2,11].

Beyond metabolic outcomes, exploratory clinical and preclinical studies have examined hydrogen administration in neurological, renal, oncologic, and immune-related contexts [1,2,52,53]. In neurological contexts, preclinical and early clinical studies suggest that molecular hydrogen may exert neuroprotective effects through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms [52,53]. A 2025 review by Wang et al. examining hydrogen therapy in Parkinson’s disease noted promising preclinical findings; however, clinical trials have thus far yielded mixed outcomes, with some reporting limited or no therapeutic benefit, highlighting the gap between preclinical promise and clinical translation [54]. Regarding renal outcomes, a 2024 perspective review by Nakayama et al., reported that electrolyzed hydrogen-rich water may reduce oxidative stress and support renal function in chronic kidney disease and dialysis patients, though the authors emphasized that evidence remains largely preclinical and that robust clinical trials are still needed [45]. A 2025 case report by Lin et al. further described improvements in renal function and reduction in chronic fatigue following molecular hydrogen therapy in an elderly patient with multiple comorbidities, including diabetic nephropathy, suggesting potential clinical relevance that warrants further investigation [55].

In oncologic and immune-related settings, hydrogen has been investigated primarily as an adjunctive intervention. A 2024 review by Zhou et al. reported that molecular hydrogen may inhibit T cell exhaustion and enhance anti-tumor immune function while mitigating oxidative stress associated with radiation and chemotherapy [56]. However, a notable mechanistic concern was raised by Hasegawa et al., who demonstrated that molecular hydrogen enhanced proliferation in four out of seven human cancer cell lines tested, suggesting that its oncologic safety profile requires careful evaluation before broader clinical application [57]. Regarding fatigue and exercise recovery, a 2024 randomized double-blind placebo-controlled crossover trial by Sládečková et al. in elite fin swimmers reported that HRW supplementation promoted muscle recovery following two strenuous training sessions performed on the same day [58]. However, not all exercise-related studies report consistent benefits; a randomized double-blind placebo-controlled crossover study by Botek et al., (2020) demonstrated unclear effects of HRW on race time and fatigue, with performance improvements observed only in slower runners, suggesting that individual baseline fitness level may modulate hydrogen’s ergogenic effects [59]. Collectively, these findings across multiple organ systems and clinical contexts support the biological activity of molecular hydrogen beyond metabolic outcomes, though evidence in most areas remains preliminary, heterogeneous, and largely insufficient to draw definitive clinical conclusions [2,11].

In oncologic and infectious disease settings, hydrogen has been investigated primarily as an adjunctive intervention, with reports describing modulation of immune exhaustion markers and oxidative stress parameters [60,61,62]. These findings remain preliminary and are often limited by small sample sizes, lack of blinding, or absence of appropriate control groups [2].

Preclinical models further support biological activity of molecular hydrogen across multiple organ systems, including neuroprotection, reproductive function, hepatic metabolism, and protection against drug-induced toxicity [1,2,63,64]. Human studies using hydrogen-rich water, saline infusion, and inhalation report benefits in metabolic syndrome, rheumatoid arthritis, fatigue and exercise recovery, inflammatory biomarkers and neuroprotection models [9,65,66]. Collectively, these studies establish safety and biological activity. While these studies provide mechanistic insight, their translational relevance to human dosing and delivery routes remains uncertain [5].

Collectively, existing clinical and preclinical evidence supports the notion that molecular hydrogen can influence redox-related biological processes [2,11]. However, the diversity of delivery methods, exposure levels, and outcome measures limits direct comparison across studies. Importantly, no conclusions can be drawn regarding the efficacy, bioavailability, or clinical utility of sublingual hydrogen delivery based on the current literature. Instead, these data primarily inform biological plausibility and help define priorities for future translational research.

4. Safety and Practical Considerations

Molecular hydrogen is generally regarded as safe when administered at concentrations well below its flammability threshold of approximately 4% in air [1,2]. Clinical studies involving hydrogen-rich water, inhalation therapy, and hydrogen-enriched dialysate have reported minimal adverse effects, with safety profiles comparable to control interventions in most trials [5,8]. These observations are consistent with hydrogen’s endogenous production by intestinal microbiota and its rapid elimination via respiration [2].

Despite this favorable safety profile, novel delivery approaches such as sublingual administration warrant careful evaluation. Key considerations include verification of dissolved hydrogen concentration at the point of delivery, assessment of hydrogen stability during storage and dispensing, and confirmation of batch-to-batch reproducibility [5]. Hydrogen is volatile, diffuses rapidly, and is lost quickly once water containers are opened. Given the rapid diffusion and dissipation of dissolved hydrogen, failure to adequately characterize these parameters could result in substantial variability in delivered dose [2]. Most protocols require large fluid volumes, bulky generators and supervised inhalation. This creates barriers for daily consistency, especially in elderly, metabolic, or mobile populations.

Measurement methodology represents an additional practical concern. While oxidation–reduction potential is sometimes used as an indirect indicator of reducing conditions, it does not reliably reflect dissolved hydrogen concentration or biological activity [5]. Accordingly, direct quantification of hydrogen content using validated analytical methods is essential for both formulation development and clinical investigation.

Finally, although hydrogen itself is non-toxic, the materials, propellants, or additives used in delivery devices must be evaluated for mucosal safety and tolerability, particularly for repeated sublingual exposure. These considerations underscore the importance of rigorous preclinical testing and quality control prior to human use [2].

5. Research Gaps and Proposed Validation Framework

Despite growing evidence supporting the biological activity and therapeutic potential of molecular hydrogen (H₂), several scientific, technological, and translational challenges continue to limit its broader clinical and industrial adoption.

5.1. Biomedical and Clinical Research Gaps

A major limitation remains the incomplete understanding of H₂’s mechanisms of action. Although numerous preclinical studies and early clinical trials have demonstrated antioxidant, anti-inflammatory, anti-apoptotic, and cytoprotective effects across cardiovascular, neurological, metabolic, and inflammatory disorders, the primary molecular targets responsible for these benefits remain incompletely defined. Current evidence implicates redox-sensitive pathways such as Nrf2, NF-κB, mitochondrial signaling, and endothelial regulatory networks; however, the relative contributions of direct radical scavenging versus indirect modulation of cellular signaling remain unclear [4,39,67].

Clinical translation is further hindered by the lack of standardized treatment protocols. Existing studies employ diverse delivery modalities including hydrogen-rich water, inhaled hydrogen gas, hydrogen-enriched saline, and emerging formulations with substantial variation in hydrogen concentration, dosing regimens, treatment duration, and outcome measures. This heterogeneity complicates cross-study comparisons, limits the strength of meta-analyses, and impedes the development of evidence-based clinical guidelines [68,69,70].

Another important gap is the limited number of large-scale randomized controlled trials. Most human studies remain exploratory, involve relatively small sample sizes, or have short follow-up periods, leaving long-term efficacy, optimal dosing strategies, and safety profiles insufficiently characterized [4,39,68,70]. Furthermore, current methods for measuring hydrogen concentrations in biological systems often lack the sensitivity and precision required to establish robust dose–response relationships. The development of advanced analytical technologies and bioprobes will therefore be essential for future pharmacokinetic and mechanistic investigations [39,71].

5.2. Materials Science and Hydrogen Storage Gaps

Significant challenges also remain in hydrogen storage and delivery technologies. Reproducibility is a persistent concern due to inconsistencies in experimental methodologies, simulation parameters, and reporting standards across studies. Greater transparency, standardized protocols, and open-access datasets have been advocated to improve research quality and facilitate independent validation [19,72].

Practical deployment represents another major obstacle. Advanced hydrogen-storage materials, including metal-organic frameworks and other porous materials, frequently demonstrate excellent storage capacity under cryogenic or high-pressure conditions but show reduced performance under ambient conditions required for real-world applications [19,73,74]. Bridging this gap between laboratory performance and practical usability remains a critical challenge for commercialization.

In addition, the enormous chemical and materials design space creates a substantial screening bottleneck. Accelerating discovery will require integration of high-throughput computational screening, machine learning approaches, and experimental validation to efficiently identify promising materials with favorable storage capacity, stability, safety, and cost-effectiveness [75,76].

5.3. Cross-Domain Challenges

Several barriers extend across both biomedical and materials science domains. Research efforts remain fragmented, with limited interdisciplinary and international collaboration. Stronger integration among basic scientists, clinicians, engineers, industry partners, and regulatory agencies will be essential to accelerate the translation of promising discoveries into clinically and commercially viable technologies [69].

Regulatory and manufacturing considerations also remain significant obstacles. The absence of harmonized regulatory frameworks, standardized quality-control measures, validated analytical methods, and scalable manufacturing protocols creates uncertainty regarding product development, safety assessment, and market approval. Addressing these challenges will be critical for ensuring reproducibility, safety, and widespread adoption of hydrogen-based technologies in both healthcare and energy sectors [69].

Future progress in molecular hydrogen research will ultimately depend on resolving mechanistic uncertainties, standardizing clinical and technological methodologies, improving hydrogen detection and storage systems, validating emerging delivery platforms such as sublingual administration, and fostering stronger interdisciplinary collaboration. Addressing these priorities will be essential to translate the growing promise of molecular hydrogen into scientifically validated therapeutic and industrial applications

5.4. Proposed Validation Framework for Sublingual Molecular Hydrogen Delivery

To advance molecular hydrogen toward novel delivery routes such as sublingual administration, a systematic translational framework is needed. Device and formulation characterization studies should first establish dissolved hydrogen concentration, stability, reproducibility, and variability under storage and dispensing conditions. Direct quantification of hydrogen content should be prioritized over surrogate measures such as oxidation–reduction potential (ORP) or pH [5,77,78]. In silico analysis shows that small changes in pH, temperature, and meter error can shift ORP more than the entire contribution of dissolved H₂, making ORP-based “H₂ meters” highly inaccurate for quantification [46]. On the other hand, direct measurement techniques including electrochemical microsensors, gas chromatography, and validated hydrogen-specific sensors are considered the gold standard for dose determination and study comparability [77,78].

Subsequently, first-in-human pharmacokinetic studies should evaluate hydrogen absorption, systemic exposure, and clearance by measuring hydrogen levels in breath, saliva, and blood following sublingual administration [66]. These investigations would determine whether sublingual delivery achieves meaningful bioavailability compared with established routes such as hydrogen-rich water or inhalation.

Pilot randomized controlled trials should then assess safety, feasibility, and biological activity using mechanistic endpoints, including oxidative stress markers, inflammatory biomarkers, endothelial function, and mitochondrial responses. Consistent with previous hydrogen research, these early studies should focus on biological effects rather than clinical efficacy claims. Additional exploratory investigations incorporating micronutrient bioavailability assessments, metabolomics, and redox-sensitive metabolic profiling may further clarify the broader physiological consequences of hydrogen-mediated redox regulation [12,39,71].

This stepwise framework from formulation characterization and pharmacokinetic evaluation to mechanistic clinical testing provides a scientifically rigorous and ethically responsible pathway for determining the feasibility and translational potential of sublingual molecular hydrogen delivery (Figure 3).

6. Future Directions

Future research on molecular hydrogen should focus on resolving several critical mechanistic, methodological, and translational gaps that currently limit its clinical integration. First, rigorous pharmacokinetic and pharmacodynamic studies are needed to determine the absorption, tissue distribution, and elimination kinetics of hydrogen across different delivery modalities, particularly for sublingual administration, which remains unexplored in peer-reviewed clinical studies. Establishing standardized dose–response relationships and exposure metrics will be essential for interpreting clinical outcomes and comparing results across studies. Second, future trials should incorporate direct quantification of dissolved hydrogen concentrations, standardized formulation protocols, and validated biomarkers of oxidative stress and inflammation to reduce the methodological heterogeneity that characterizes the current literature. Mechanistic investigations should further clarify the relative contributions of direct radical interactions versus modulation of redox-sensitive signaling pathways, including Nrf2, NF-κB, mitochondrial function, and endothelial signaling networks. In parallel, well-designed randomized controlled trials with adequate sample sizes and clearly defined endpoints are necessary to evaluate clinical efficacy across conditions associated with oxidative and inflammatory stress. Finally, emerging areas such as precision redox medicine, interactions with the gut microbiome, and the development of stable hydrogen delivery technologies may expand the therapeutic potential of hydrogen while improving reproducibility and translational relevance. Holistically, addressing these priorities will be essential to determine whether molecular hydrogen can move from a promising redox modulator to a clinically validated therapeutic strategy.

7. Conclusions

Molecular hydrogen has emerged as a biologically active molecule with distinctive physicochemical properties and the capacity to influence redox-related processes. Experimental and clinical studies using established delivery routes, including hydrogen-rich water and inhalation, suggest that hydrogen can modulate oxidative stress, inflammatory responses, and redox-sensitive signaling pathways such as Nrf2 and NF-κB. These effects appear to occur without broad suppression of physiological reactive oxygen species, supporting the concept of hydrogen as a selective redox modulator rather than a conventional antioxidant.

Despite growing interest and an expanding evidence base, current human data remain heterogeneous, and definitive conclusions regarding clinical efficacy are limited. Importantly, sublingual delivery of molecular hydrogen has not yet been evaluated in peer-reviewed studies. While physicochemical considerations provide a plausible rationale for exploring this route, its feasibility, bioavailability, and biological relevance must be established through rigorous formulation characterization, pharmacokinetic assessment, and well-controlled clinical trials.

Future research should prioritize methodological rigor, transparent reporting of hydrogen concentrations, and mechanistically grounded outcome measures. Until such data are available, claims regarding specific therapeutic indications or advantages of novel delivery routes should be regarded as investigational. Within these constraints, molecular hydrogen continues to represent an intriguing subject in redox biology, warranting careful and systematic investigation.