Melatonin and DNA Integrity: The Impact of Exogenous Administration in Exercise-Induced Oxidative Stress—A Systematic Review

Abstract

Background/Objectives: Intense physical exercise leads to oxidative stress, causing cellular and DNA damage in athletes. Melatonin (MLT), a hormone with antioxidant and anti-inflammatory properties, is increasingly used to counteract these effects. However, its specific role in protecting DNA integrity and modulating repair mechanisms post-exercise remains unclear. This systematic review aimed to synthesize clinical evidence on the effects of exogenous MLT supplementation in reducing exercise-induced oxidative stress, reducing DNA damage, and influencing DNA integrity in healthy, physically active individuals. Methods: A comprehensive search was conducted in PubMed and Scopus up to 25 March 2025, for randomized or controlled clinical trials assessing exogenous MLT in healthy, physically active adults, with outcomes related to oxidative stress, DNA damage, or DNA repair. Risk of bias was evaluated using the RoB2 tool. Due to heterogeneity in study designs and outcomes, results were synthesized narratively. Results: Six clinical trials met the inclusion criteria, with MLT administered as a single dose (6–10 mg) or in repeated doses over 6 days to 4 weeks. Across the studies, MLT consistently reduced oxidative stress markers (malondialdehyde, advanced oxidation protein products), muscle damage indicators (creatine kinase, LDH), and inflammation, while increasing antioxidant enzyme activity (SOD, GPx). Only one study directly assessed DNA damage, reporting significantly reduced DNA fragmentation (comet assay) in the MLT group compared to placebo. No studies directly evaluated DNA repair pathways. Conclusions: Exogenous MLT supplementation appears effective in attenuating exercise-induced oxidative stress and may reduce DNA damage in athletes. While findings support its antioxidant and cytoprotective roles, further rigorous trials are needed to clarify its direct effects on DNA repair mechanisms in sports medicine. Funding: This review received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Registration: This review was prospectively registered in the PROSPERO database (CRD420231039805).

1. Introduction

Regular physical activity represents a cornerstone of health promotion, conferring multisystemic benefits ranging from cardiovascular optimization to metabolic regulation and neuroplasticity enhancement [1,2,3]. However, this ostensibly beneficial intervention harbors a physiological paradox—when exercise intensity or duration exceeds certain thresholds, the very metabolic processes that sustain life can precipitate a state of oxidative imbalance that potentially compromises cellular integrity [4,5,6]. This multifaceted phenomenon of exercise-induced oxidative stress has garnered considerable scientific attention, particularly regarding its impact on genomic stability and the potential protective role of endogenous and exogenous antioxidants such as N-acetyl-5-methoxytryptamine (Melatonin) [7,8]. To comprehensively evaluate the physiological stress associated with strenuous exercise and interventions aimed at mitigating its deleterious effects, researchers have established a sophisticated biomarker framework encompassing biochemical indicators, oxidative stress markers, and DNA damage/repair parameters [9,10].

Emerging studies highlight urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and γH2A.X as critical biomarkers for assessing oxidative DNA damage repair capacity in Melatonin (MLT) supplementation research [11,12,13]. 8-OH-dG, a byproduct of guanine oxidation, reflects systemic DNA repair efficiency, while γH2A.X serves as a sensitive marker of double-strand breaks. Recent advances in omics technologies, including metabolomics and proteomics, have enabled comprehensive profiling of MLT’s regulatory effects on oxidative stress pathways. For instance, proteomic analyses reveal MLT-mediated upregulation of antioxidant enzymes like superoxide dismutase and glutathione peroxidase, while metabolomic studies identify shifts in redox-related metabolites such as glutathione and reactive oxygen species (ROS) scavengers [3,12]. These multi-omics approaches provide mechanistic insights into MLT’s capacity to enhance DNA repair systems under exercise-induced oxidative stress, though lipidomic investigations remain underrepresented in the current literature.

1.1. Physiological Mechanisms of Exercise-Induced Oxidative Stress

The genesis of oxidative stress during intense physical exertion is intrinsically linked to profound metabolic alterations occurring within active skeletal muscles. When exercise intensity surpasses approximately 60–70% of maximal oxygen consumption (VO₂ max), ATP demand increases exponentially to fuel repeated cycles of actin-myosin cross-bridge formation and maintain ion homeostasis via membrane pumps [5,14,15]. Oxygen consumption in working myocytes can escalate dramatically—potentially 100-fold above resting values in specific muscle fibers—with the electron transport chain (ETC) initially attempting to accommodate this heightened energetic demand through oxidative phosphorylation [1].

However, as exercise intensity transcends the anaerobic threshold, the ETC’s capacity for efficient electron processing becomes overwhelmed. This metabolic crisis prompts a compensatory shift toward anaerobic glycolysis, accelerating the breakdown of muscle glycogen stores and leading to the rapid production of pyruvate [3,16,17]. When pyruvate generation outpaces mitochondrial oxidative capacity, conversion to lactate ensues, with concurrent H⁺ accumulation contributing to intracellular acidosis. This acidotic environment, coupled with exercise-induced hyperthermia, further exacerbates reactive oxygen species (ROS) production and potentially compromises antioxidant enzyme function [1,17,18].

The intensified metabolic flux through mitochondria during strenuous exercise creates considerable oxidative stress on the ETC. Despite its remarkable efficiency, approximately 2–5% of electrons traversing the respiratory chain prematurely escape—primarily from Complexes I and III—and react directly with molecular oxygen to form superoxide radical (O₂⁻-), the primary ROS generated mitochondrially [1,3,4,19]:

$${\mathrm{O}}_2+{\mathrm{e}}^{-}\to {\mathrm{O}}_2^{·-}$$

Simultaneously, ATP hydrolysis during intensive muscular work activates the purine nucleotide cycle, leading to hypoxanthine accumulation. In microdomains experiencing relative ischemia, xanthine oxidase (XO) catalyzes hypoxanthine oxidation to uric acid, generating both O₂⁻- and hydrogen peroxide (H₂O₂) as byproducts. Further contributing to the oxidative burden are NADPH oxidases (NOX), particularly NOX2 isoforms localized to sarcolemma and sarcoplasmic reticulum, which are activated by contractile activity and calcium-dependent signaling cascades [3,4,20,21].

The superoxide radical from these multiple sources undergoes dismutation—catalyzed by superoxide dismutase (SOD) enzymes—forming the less reactive but more diffusible H₂O₂:

$$2{\mathrm{O}}_2^{·-}+2{\mathrm{H}}^{+}\stackrel{\mathrm{S}\mathrm{O}\mathrm{D}}{\to }{\mathrm{H}}_2{\mathrm{O}}_2+{\mathrm{O}}_2$$

In biological systems, H₂O₂ participates in the iron-catalyzed Haber-Weiss reaction, the physiologically relevant pathway for hydroxyl radical generation during exercise. Unlike classical Fenton chemistry, intracellular iron exists bound to proteins or as chelated complexes in the labile iron pool. This iron catalyzes the two-step process: superoxide reduces Fe³⁺ to Fe²⁺, followed by Fe²⁺ reacting with H₂O₂ in a Fenton-type reaction [22,23,24,25].

O₂⁻· + H₂O₂ → ·OH + OH⁻ + O₂

This net reaction represents the major pathway for hydroxyl radical production in exercising muscle, particularly in microdomains where exercise-induced metabolic changes may transiently increase iron availability from storage proteins [22,23].

Concurrently, elevated nitric oxide (NO⁻) production during exercise, particularly via endothelial nitric oxide synthase (eNOS) activation promoting exercise-induced vasodilation, can react with superoxide at diffusion-limited rates to form peroxynitrite (ONOO⁻):

$${\mathrm{O}}_2^{·-}+{\mathrm{N}\mathrm{O}}^{·}\to {\mathrm{O}\mathrm{N}\mathrm{O}\mathrm{O}}^{-}$$

This collective burden from multiple ROS/RNS (reactive oxygen species/reactive nitrogen species) sources can temporarily overwhelm endogenous antioxidant defenses, initiating oxidative damage cascades [1,17,26].

1.2. Comprehensive Biomarker Profiling of Stress Responses to Exercise

During and after high-intensity or prolonged exercise, the assessment of physiological stress and cellular integrity relies on a multidimensional biomarker approach. Biochemical biomarkers such as creatine kinase (CK), lactate dehydrogenase (LDH), and hepatic enzymes—like Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT)—are widely recognized indicators of tissue injury, metabolic stress, and organ function, reflecting acute muscle damage, hepatic strain, and renal workload in response to strenuous physical activity [9,27]. In parallel, ROS oxidation biomarkers provide direct insight into the redox status of the organism. These include products of lipid peroxidation (malondialdehyde, MDA), protein oxidation (advanced oxidation protein products, AOPP), and the activity or capacity of endogenous antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx) [3,28]. Changes in these markers reveal the balance between reactive oxygen species generation and the efficacy of antioxidant defenses, serving as sensitive indicators of oxidative stress and its modulation by interventions such as MLT supplementation. Finally, DNA repair biomarkers—including direct measures such as the comet assay (quantifying DNA strand breaks in lymphocytes) and specific oxidative DNA lesions (e.g., 8-hydroxy-2′-deoxyguanosine, 8-OHdG), as well as the activity of repair enzymes like OGG1 and APE1—capture the genotoxic consequences of exercise-induced ROS and the cellular capacity for genomic maintenance and repair [29,30,31]. Together, this integrated biomarker framework enables a comprehensive evaluation of exercise-induced stress, the efficacy of antioxidant interventions, and the preservation of cellular and genomic integrity in athletes [29,32,33].

These biomarkers are mechanistically linked to glycogen metabolism during exercise, as depicted in Figure 1. When exercise intensity exceeds 60–70% of VO₂ max, rapid glycogen depletion occurs as pyruvate production increases. This pyruvate faces two metabolic fates: anaerobic conversion to lactate via LDH or aerobic metabolism through the pyruvate dehydrogenase complex (PDHC) to form acetyl-CoA for entry into the Krebs cycle [16,34,35,36]. The intensified electron flow through the electron transport chain (ETC) significantly increases the probability of electron leakage, primarily at Complexes I and III, generating superoxide radical according to the reaction [4,35,37]:

$${\mathrm{O}}_2+{\mathrm{e}}^{-}\to {\mathrm{O}}_2^{·-}$$

1.2.1. Biochemical Biomarkers of Exercise-Induced Cellular Damage and Metabolic Stress

Biochemical biomarkers represent the first tier in the assessment framework, providing insights into tissue damage, energetic status, and systemic metabolic alterations induced by strenuous exercise [4,5,38,39]. These include:

Creatine Kinase (CK): A cytosolic enzyme predominantly found in muscle tissue, CK catalyzes the reversible phosphorylation of creatine by ATP, maintaining energetic homeostasis. Exercise-induced disruption of sarcolemmal integrity facilitates CK leakage into circulation, with isoenzyme analysis providing insights into the specific tissues affected. Studies consistently demonstrate significant post-exercise CK elevations, particularly following eccentric or high-intensity exercise, with serum concentrations remaining elevated for 24–72 h [8,40].

Lactate Dehydrogenase (LDH): This ubiquitous enzyme catalyzes the interconversion of pyruvate and lactate, serving as a key regulator of anaerobic metabolism and cellular redox balance. Like CK, exercise-induced membrane perturbation facilitates LDH release into circulation, with isoenzyme patterns (LDH1-5) potentially distinguishing between cardiac, hepatic, and skeletal muscle origins of injury [8,34,38].

Hepatic Enzymes: AST (aspartate aminotransferase), ALT (alanine aminotransferase) and GGT (gamma-glutamyl transferase) are primarily regarded as markers of liver function, but their presence and activity also indicate broader systemic stress and potential damage to extrahepatic tissues. AST is found in both cytosolic and mitochondrial forms, while ALT is predominantly cytosolic; both play crucial roles in amino acid metabolism and gluconeogenesis. Transient increases in AST and ALT following exercise may signal hepatocellular stress and, due to their distribution in muscle, can also reflect skeletal muscle injury. GGT, in addition to its established role in liver and biliary tract health, is recognized as a sensitive marker of oxidative stress and has been associated with systemic inflammation and metabolic disturbances, further supporting its utility as an indicator of overall physiological stress

Renal Function Markers (Creatinine, Urea): These nitrogenous compounds reflect both glomerular filtration rate and protein catabolism. Transient post-exercise elevations may indicate reduced renal perfusion during intense activity and accelerated protein turnover [41,42].

Metabolic Substrates (Glucose, Lactate): Direct measures of energetic intermediates that reflect the balance between glycolysis, gluconeogenesis, and oxidative metabolism. Exercise-induced alterations in these parameters provide insights into substrate utilization patterns and metabolic efficiency [16,43,44].

The mechanistic relationship between these biochemical markers and exercise intensity follows a threshold pattern, with significant elevations typically observed when glycogen depletion and cellular membrane perturbation coincide with redox imbalance, as illustrated later in Figure 1 [45].

1.2.2. Biomarkers of Oxidative Stress: Assessing Redox Imbalance and Antioxidant Response

The second tier of assessment focuses on direct markers of oxidative damage and antioxidant capacity, providing mechanistic insights into redox perturbations induced by strenuous exercise [8,32]:

Lipid Peroxidation Products: Polyunsaturated fatty acids within cellular membranes are primary ROS targets, undergoing peroxidation that generates reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [28,44]. These products form through a self-propagating chain reaction initiated when hydroxyl radicals abstract hydrogen atoms from lipid molecules:

$$\begin{array}{c}{\mathrm{L}}^{·}+{\mathrm{O}}_2\to {\mathrm{L}\mathrm{O}\mathrm{O}}^{·}\\ {\mathrm{L}\mathrm{O}\mathrm{O}}^{·}+\mathrm{L}\mathrm{H}\to \mathrm{L}\mathrm{O}\mathrm{O}\mathrm{H}+{\mathrm{L}}^{·}\end{array}$$

MDA, quantified via thiobarbituric acid reactive substance (TBARS) assay or more specific HPLC methods, serves as a widely utilized biomarker for exercise-induced oxidative stress, with concentrations typically increasing 15–40% following high-intensity exercise [46,47,48].

Protein Oxidation Markers: ROS-induced protein modifications include carbonylation, thiol oxidation, and tyrosine nitration. Advanced oxidation protein products (AOPP), formed primarily through chlorinated oxidant action on protein lysine residues, represent a clinically relevant marker of protein oxidation. AOPP concentrations demonstrate significant post-exercise elevations, correlating with exercise intensity and duration [8,42].

Nitric Oxide Derivatives: Exercise-induced alterations in vascular and cellular NO metabolism generate nitrite/nitrate (NOx) and potentially nitrotyrosine-containing proteins. These markers reflect the complex interplay between NO signaling, vasodilation, and potential peroxynitrite formation during intense exercise [4,47,49].

Antioxidant Enzymes: The activity and expression of key antioxidant enzymes, including SOD isoforms (cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), provide insights into the adaptive capacity of endogenous defense systems [4,20,44]. These enzymes catalyze critical reactions in ROS detoxification:

$$\begin{array}{c}2{\mathrm{H}}_2{\mathrm{O}}_2\stackrel{\mathrm{C}\mathrm{A}\mathrm{T}}{\to }2{\mathrm{H}}_2\mathrm{O}+{\mathrm{O}}_2\\ {\mathrm{H}}_2{\mathrm{O}}_2+2\mathrm{G}\mathrm{S}\mathrm{H}\stackrel{\mathrm{G}\mathrm{P}\mathrm{x}}{\to }\mathrm{G}\mathrm{S}\mathrm{S}\mathrm{G}+2{\mathrm{H}}_2\mathrm{O}\end{array}$$

Exercise training typically enhances antioxidant enzyme expression through hormetic adaptations, whereas acute exhaustive exercise may temporarily reduce enzyme activity through oxidative inactivation or substrate depletion [1,3,17].

Global Antioxidant Capacity: Integrated measures such as oxygen radical absorption capacity (ORAC) or total antioxidant capacity (TAC) reflect the collective action of enzymatic and non-enzymatic antioxidants, providing a holistic assessment of systemic redox buffering capacity [8,50].

1.2.3. Molecular Markers of DNA Repair Pathways and Genomic Integrity

The third tier of assessment focuses on DNA damage and repair processes, representing the most concerning aspect of exercise-induced oxidative stress due to potential mutagenic and cytotoxic consequences [10,29,51]:

Comet Assay (Single Cell Gel Electrophoresis): This versatile technique visualizes and quantifies DNA strand breaks in individual cells, typically lymphocytes. The migration of DNA fragments during electrophoresis creates a “comet tail,” with parameters such as tail length, intensity, and moment correlating with damage severity. Studies utilizing the alkaline comet assay consistently demonstrate transient increases in lymphocyte DNA damage following exhaustive exercise, with recovery typically observed within 24–72 h in adequately trained individuals [14,44,52,53].

Oxidative DNA Lesions: Among numerous oxidative DNA modifications, 8-hydroxy-2′-deoxyguanosine (8-OHdG) represents the most extensively characterized. Formed when hydroxyl radicals attack guanine’s C-8 position, 8-OHdG is highly mutagenic due to its tendency to mispair with adenine during replication. Urinary 8-OHdG excretion reflects the integrated processes of oxidative DNA damage and repair efficiency, with exercise-induced elevations typically proportional to intensity and duration [4,54].

DNA Repair Enzyme Activity: The base excision repair (BER) pathway represents the primary mechanism for addressing oxidative DNA lesions [10,31,51,55]. Key enzymes in this pathway include:

• OGG1 (8-oxoguanine DNA glycosylase): Recognizes and excises 8-OHdG, initiating the repair process [47].
• APE1 (apurinic/apyrimidinic endonuclease 1): Cleaves the DNA backbone at abasic sites generated by glycosylases [56].
• XRCC1, DNA polymerase β, and DNA ligase III: Complete the repair process through end processing, gap filling, and strand ligation [29,56].

Assessment of repair enzyme expression, activity, or polymorphisms provides insights into individual susceptibility to exercise-induced genomic instability [29,32,33]. Among these enzymes, poly(ADP-ribose) polymerase (PARP) plays a particularly pivotal role: as an NAD⁺-dependent enzyme, PARP is essential for recognizing DNA damage and orchestrating the repair process. However, individuals with heightened PARP activity or genetic predispositions may be more vulnerable to the consequences of extensive DNA damage, as overactivation of PARP can significantly deplete cellular NAD⁺ levels. This depletion may impair energy metabolism and further intensify the metabolic stress associated with exercise [35,47,57].

1.3. Biomarkers of Oxidative Stress: Assessing Redox Imbalance and Antioxidant Response

Biomarkers of oxidative stress can be broadly categorized as direct or indirect, reflecting distinct biological processes and methodological approaches. Direct biomarkers are those that measure reactive oxygen species (ROS) or reactive nitrogen species (RNS) themselves, or their immediate products, within biological samples. These include techniques such as fluorogenic probes for superoxide, hydrogen peroxide, or hydroxyl radicals, which provide a snapshot of the acute redox status in cells or tissues. However, due to the high reactivity and short half-lives of these species, direct measurements can be technically challenging and may not fully capture the cumulative oxidative burden experienced during or after exercise. In contrast, indirect biomarkers assess the downstream consequences of oxidative stress by quantifying stable end products of macromolecular damage—such as malondialdehyde (MDA) for lipid peroxidation, protein carbonyls (PC) for protein oxidation, or 8-hydroxy-2′-deoxyguanosine (8-OHdG) for DNA oxidation—as well as changes in antioxidant defenses, including the activity of enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Indirect markers thus provide an integrated view of oxidative stress over time, reflecting both the extent of molecular injury and the adaptive capacity of endogenous defense systems. The distinction between direct and indirect biomarkers is essential for interpreting redox dynamics in exercise physiology, as each offers complementary insights into the mechanisms and consequences of oxidative stress in response to physical activity [11,22,58,59].

In the context of the systematic review on MLT and exercise-induced oxidative stress, biomarkers are classified as direct or indirect based on their ability to measure reactive oxygen species (ROS) or their immediate effects versus downstream consequences of oxidative damage [2,39,45,60]. This classification is critical for interpreting the redox dynamics in exercise physiology and aligns with established frameworks in the literature.

• Direct Biomarkers measure ROS or reactive nitrogen species (RNS) directly or their immediate products, providing a real-time snapshot of acute oxidative stress. Examples include fluorogenic probes for superoxide, hydrogen peroxide, or hydroxyl radicals. However, due to the short half-life and high reactivity of ROS, direct measurements are technically challenging and often fail to reflect cumulative oxidative burden during exercise [11,22,58,59].
• Indirect Biomarkers assess the downstream effects of oxidative stress by measuring stable end-products of macromolecular damage or changes in antioxidant defenses, offering an integrated view of oxidative stress over time [61]. In the included studies, indirect biomarkers were predominant and encompassed several categories:
  • Biochemical Biomarkers: Indicators such as creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea reflect muscle damage, metabolic stress, and organ function. These are indirect as they signal systemic stress and cellular injury resulting from ROS overproduction rather than measuring ROS themselves [62].
  • ROS Oxidation Biomarkers: Markers like malondialdehyde (MDA) for lipid peroxidation, advanced oxidation protein products (AOPP) for protein oxidation, and antioxidant enzyme activities (e.g., superoxide dismutase [SOD], glutathione peroxidase [GPx], glutathione reductase [GR]) were frequently assessed. These are indirect because they measure the consequences of ROS activity or the body’s adaptive response rather than ROS directly [49,62].
  • Other Indirect Markers: Total antioxidant capacity (TAC), oxygen radical absorbance capacity (ORAC), lipid peroxidation (LPO), nitric oxide metabolites (NOx), reduced glutathione (GSH), and oxidized glutathione (GSSG) were also reported, reflecting systemic redox balance and oxidative damage indirectly [62].

Biochemical Rationale and Clinical Correlation

Direct Biomarkers such as the comet assay provide immediate evidence of DNA strand breaks, directly measuring genomic damage resulting from exercise-induced reactive oxygen species (ROS). For example, in the study by Ortiz-Franco et al., the application of the comet assay established a direct link between oxidative stress and DNA integrity, underscoring the clinical relevance of this direct biomarker for assessing MLT’s genoprotective potential in athletes.

In contrast, Indirect Biomarkers like malondialdehyde (MDA) and advanced oxidation protein products (AOPP) reflect the cumulative oxidative damage to lipids and proteins, respectively. These markers correlate with systemic oxidative stress that may exacerbate DNA damage over time. Additionally, the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) serves as an indirect biomarker by indicating the body’s adaptive response to increased ROS, thereby supporting DNA protection through the reduction in oxidative load. Clinically, observed reductions in creatine kinase (CK) and lactate dehydrogenase (LDH) following MLT supplementation further support the utility of indirect biomarkers, as they suggest decreased muscle damage and a consequent reduction in secondary oxidative stress that could otherwise compromise DNA stability.

This classification underscores the complementary nature of direct and indirect biomarkers in evaluating exercise-induced oxidative stress. While direct measures like the comet assay are critical for specific outcomes such as DNA damage, indirect markers provide a broader perspective on systemic redox status, essential for understanding MLT’s multifaceted protective effects in sports medicine.

1.4. Melatonin as a Pleiotropic Antioxidant in the Context of Exercise-Induced Oxidative Stress

MLT (N-acetyl-5-methoxytryptamine) has emerged as a compound of significant interest in mitigating exercise-induced oxidative stress and its consequences. Although primarily synthesized by the pineal gland following a circadian rhythm, MLT is also produced in various peripheral cells and organs, with mitochondria themselves acting as important intracellular sites of its synthesis, MLT’s functions extend far beyond sleep regulation [39,63]. Its amphipathic molecular structure facilitates penetration of cellular and subcellular membranes, including the blood–brain barrier and mitochondrial membranes, allowing access to compartments where conventional antioxidants may have limited distribution [8,29,39,63]. MLT intervenes at multiple points in the exercise-induced metabolic and oxidative cascade. At the substrate level, MLT modulates pyruvate metabolism while enhancing β-oxidation through peroxisome proliferator-activated receptor (PPAR) activation, potentially preserving glycogen stores during exercise [64,65]. The mechanistic interactions between exercise-induced metabolic flux, mitochondrial ROS generation, antioxidant defenses, and the multifaceted protective actions of MLT are summarized in Figure 1.

At the mitochondrial level, MLT targets Complex III of the ETC, significantly reducing electron leakage and subsequent superoxide formation [28,44]. This action directly attenuates the initiation of oxidative damage cascades that generate lipid peroxyl radicals in a chain reaction:

$$L^{·}+O_2\to {LOO}^{·}{LOO}^{·}+LH\to LOOH+L^{·}$$

MLT’s impact on the three biomarker categories is comprehensive and mechanistically interlinked:

Biochemical Biomarkers: MLT supplementation consistently reduces exercise-induced elevations in muscle damage markers (CK, LDH) and hepatic enzymes (AST, ALT), suggesting enhanced membrane stability and reduced tissue injury. For instance, Farjallah et al. observed a 45% reduction in CK levels following MLT supplementation in athletes undergoing intensive training [7].

ROS Oxidation Biomarkers: MLT significantly attenuates lipid and protein oxidation while enhancing antioxidant enzyme activity. Studies demonstrate reduced MDA (23–40% decrease) and AOPP levels following MLT administration, concurrent with increased SOD and GPx activity [8,45].

DNA Repair Biomarkers: Most intriguingly, MLT appears to influence DNA integrity through multiple complementary mechanisms. By reducing primary ROS production, MLT preventatively diminishes DNA damage burden. Additionally, and perhaps more importantly, MLT activates the Base Excision Repair (BER) pathway through enhanced OGG1 and APE1 activity [42,49,51], as shown in Figure 1. This activation ensures more efficient removal of oxidized DNA lesions like 8-OHdG and repair of DNA strand breaks. The comet assay has demonstrated significantly reduced DNA fragmentation in lymphocytes from MLT-supplemented subjects compared to placebo controls following high-intensity exercise [29,32,33].

Beyond its antioxidant effects, MLT exhibits anti-inflammatory properties through inhibition of NF-κB signaling and reduction in pro-inflammatory cytokines (TNF-α, IL-6), as depicted in the lower portion of Figure 1. This anti-inflammatory action helps attenuate secondary oxidative damage and explains the reduction in muscle damage biomarkers observed following MLT supplementation in exercising subjects [7,42,66,67].

The recovery-enhancing effects of MLT are particularly significant for athletes engaging in multiple training sessions or competition phases. By potentiating exercise-induced increases in skeletal muscle PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha), MLT optimizes glycogen replenishment during the recovery phase [68,69]. This molecular adaptation facilitates more efficient energy substrate restoration and potentially enhances subsequent exercise performance.

Despite growing evidence supporting MLT protective effects in exercise physiology, critical knowledge gaps persist regarding optimal dosing regimens, mechanistic pathways, and potential counterproductive interactions. The marked heterogeneity in supplementation protocols—exemplified by dose variations (5–100 mg/day) and inconsistent administration timing relative to exercise sessions—hinders both data interpretation and clinical translation [3]. Emerging research indicates that concurrent dietary antioxidants (polyphenols, vitamins C/E) demonstrate independent oxidative stress mitigation properties while potentially engaging in synergistic or antagonistic interactions with MLT supplementation [70]. Furthermore, sleep parameters exhibit bidirectional relationships with endogenous MLT secretion through circadian regulation mechanisms, creating complex modulatory effects on baseline antioxidant capacity and exogenous MLT responsiveness [71]. Although MLT’s antioxidant effects on oxidative biomarkers and muscle damage markers are well-established, its influence on DNA repair enzymes has been primarily characterized in non-exercise models and preclinical studies, leaving their translational relevance to human exercise physiology underexplored [35,47,57].

Additionally, emerging evidence suggests a potentially hormetic relationship between ROS signaling and exercise adaptation, raising questions about whether indiscriminate ROS suppression might attenuate beneficial training responses [1,3,17]. This creates a nuanced risk-benefit scenario where optimal antioxidant intervention may require precise targeting and timing to balance acute protection against long-term adaptations.

The objective of this comprehensive review is to critically examine the current scientific literature surrounding MLT’s role in mitigating exercise-induced oxidative stress and DNA damage, also considering practical aspects such as optimal dosing regimens, timing of administration, safety profiles, and potential interactions with exercise-induced adaptations. By systematically reviewing the literature on biochemical, oxidative, and genomic biomarkers, we aim to critically evaluate and synthesize the experimental evidence regarding MLT’s potential protective effects. We aim to provide a mechanistic framework for understanding MLT’s protective effects and guide evidence-based implementation strategies for both athletic performance and long-term health preservation.

2. Materials and Methods

2.1. PROSPERO Registration

This systematic review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines (Supplementary Table S1: PRISMA Methodological Documentation). The protocol was prospectively registered in PROSPERO (registration number: 1039805). Details in Supplementary Table S2: PROSPERO Methodological Documentation. Prospective registration in PROSPERO ensure public disclosure of the review’s objectives, methods, and inclusion/exclusion strategies prior to data collection and analysis, thereby enhancing the study’s credibility and facilitating reproducibility by other researchers. Furthermore, protocol registration enables the monitoring of any methodological deviations throughout the review process, thus supporting scientific integrity and the reliability of the reported findings, as recommended by PRISMA guidelines [72] and leading high-impact publications in systematic review and meta-analysis methodology.

2.2. Search Strategy

This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, ensuring transparency and reproducibility throughout the research process. A comprehensive literature search was conducted across two major databases—PubMed (which includes MEDLINE and additional resources) and Scopus—to identify studies examining the interplay between MLT, oxidative DNA damage, and exercise [73]. The search was restricted to publications from the past ten years, up to 25 March 2025, and included articles in English, Spanish, or Portuguese. Search Strings Used:

• PubMed: “melatonin” [MeSH Terms] OR “melatonin” [All Fields] OR “melatonin’s” [All Fields] OR “melatonine” [All Fields] OR “melatonins” [All Fields]
• Scopus: (melatonin OR n-acetyl-5-methoxytryptamine) AND oxidative AND dna AND (damage OR repair) AND (exercise OR exercises)

This systematic approach ensured a thorough and reproducible search for evidence pertinent to the review’s objectives [72,74,75].

2.3. Study Selection

All references retrieved from the database search were managed using Zotero [75], an open-source reference management software, which facilitated the identification and removal of duplicates based on DOI and author information. The initial screening excluded non-original research formats, such as letters to the editor, editorial comments, conference abstracts, and case reports. Studies involving participants with clinical comorbidities were also excluded to minimize confounding factors related to baseline oxidative stress and DNA repair capacity. A comprehensive list of exclusion criteria is detailed in Supplementary Table S3: Comprehensive Exclusion Criteria.

Only original research articles that provided sufficient methodological detail for data extraction and quality assessment were considered eligible, with no restrictions on publication year. The screening process was conducted independently by two reviewers (V.B. and K.S.L.), who evaluated titles, abstracts, and full texts. Any disagreements were resolved by consensus, or, if necessary, by a third reviewer (N.M.M.G.). This rigorous approach ensured the inclusion of studies directly relevant to the review’s objective and enhanced the transparency and reproducibility of the selection process.

2.4. Inclusion and Exclusion Criteria

The inclusion and exclusion criteria have been developed based on the study’s aim to evaluate the impact of exogenous MLT administration on exercise-induced oxidative stress and DNA repair mechanisms. These criteria follow the PICOS framework (Population, Intervention, Comparator, Outcomes, and Study Type) to ensure the relevance and consistency of the selected studies [72] (

Table 1. Structured PICO* Table Based on Inclusion and Exclusion Criteria.

MeSH/Descritores	Description/Definition in Study	Component
“Athletes”, “Exercise”, “Healthy Volunteers”, “Oxidative Stress”, “DNA Damage”	Healthy humans undergoing intense physical exercise protocols capable of inducing oxidative stress and DNA damage, including athletes or physically active individuals, without age or gender restrictions, if they do not have significant medical comorbidities.	P (Population)
“MLT”, “Dietary Supplements”, “Administration, Oral”, “Antioxidants”	Exogenous administration of MLT, regardless of dose, route of administration (oral or other), pharmaceutical form, or timing of administration (before, during, or after exercise).	I (Intervention)
“Placebo”, “Control Groups”, “No Intervention”	Control groups that received placebo or no MLT supplementation, allowing direct comparison of the effects of the intervention.	C (Comparator)
	Studies that assessed at least one of the following outcomes:	O (Outcomes)
“8-Hydroxy-2′-Deoxyguanosine”, “Comet Assay”, “DNA Breaks”	- Oxidative DNA damage Direct biomarkers such as 8-hydroxy-2′-deoxyguanosine (8-OHdG), comet assay, or DNA strand breaks.
“DNA Repair”, “Base Excision Repair”, “Enzymes”	- DNA repair mechanisms Expression of genes related to repair or repair enzyme activity (e.g., via BER).
“Oxidative Stress”, “Malondialdehyde”, “Superoxide Dismutase”, “Glutathione Peroxidase”	- Oxidative stress Systemic or local indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), or other antioxidant markers.

* This table outlines the PICO (Population, Intervention, Comparator, Outcomes) framework used to define the eligibility criteria for studies included in the systematic review on the effects of exogenous MLT (MLT) supplementation on exercise-induced oxidative stress and DNA repair. Each component specifies the targeted demographics, intervention characteristics, control conditions, and relevant outcomes, ensuring methodological coherence and alignment with PRISMA 2020 guidelines. MeSH (Medical Subject Headings) and DeCS (Health Sciences Descriptors) terms are provided to reflect the standardized vocabulary used in the literature search. The framework facilitates a focused synthesis of evidence, highlights research gaps such as limited data on DNA repair biomarkers, and serves as a guide for standardizing future research protocols in exercise and redox biology. Detailed lists of included and excluded studies are available in Supplementary Table S3: Comprehensive Exclusion Criteria.

).

• Population: Focused Demographics and Health Status
  The Population component targets healthy humans undergoing intense physical exercise, excluding individuals with comorbidities (e.g., diabetes, cardiovascular diseases). This exclusion is biologically justified, as chronic conditions alter baseline redox homeostasis and DNA repair capacity through persistent inflammation, mitochondrial dysfunction, or metabolic dysregulation. Such confounders could obscure the transient oxidative perturbations induced by acute exercise, undermining the assessment of MLT-specific effects. By restricting the population to athletes or active individuals without comorbidities, the review isolates exercise-induced oxidative stress as the primary variable, ensuring internal validity and translational relevance to sports medicine.
• Intervention: Standardization of MLT Supplementation
  The Intervention criterion mandates exogenous MLT administration across doses (5–100 mg), routes (oral/other), and timing (pre-/post-exercise). This flexibility accommodates variability in existing literature while maintaining focus on MLT’s pharmacodynamic actions. Excluding studies combining MLT with other antioxidants ensures the specificity of findings, as synergistic or antagonistic interactions could complicate mechanistic interpretations. For example, co-administration with vitamin C or E might amplify antioxidant effects but obscure MLT’s direct role in DNA repair pathways. By prioritizing standalone MLT interventions, the review clarifies its therapeutic potential in exercise contexts.
• Comparator: Ethical and Methodological Rigor
  The Comparator requirement for placebo or no-intervention control groups minimizes bias by isolating MLT’s effects from placebo responses or natural recovery processes. Uncontrolled designs were excluded because they preclude causal attribution, a critical limitation given the multifactorial nature of oxidative stress. Placebo-controlled trials, such as those using lactose or cellulose capsules, ensure blinding integrity and ethical feasibility, as MLT’s safety profile permits its use in athletic populations without significant risks.
• Outcomes: Biomarker Standardization and Mechanistic Insights
  Outcomes were restricted to direct biomarkers of oxidative DNA damage (8-OHdG, comet assay), DNA repair indicators (BER enzyme activity), and systemic oxidative stress markers (MDA, SOD, GPx). This tripartite focus ensures a comprehensive evaluation of MLT’s dual role in preventing damage and enhancing repair. Exclusion of studies lacking quantitative biomarker data or employing non-validated methods (e.g., subjective fatigue scales) strengthens the review’s analytical robustness. For instance, the comet assay’s inclusion in only one study underscores the need for standardized DNA damage assessments in future research, a gap highlighted in the discussion.

The PICO framework’s integration ensures methodological coherence, enabling a focused synthesis of evidence on MLT’s antioxidant and genoprotective roles. By harmonizing inclusion/exclusion criteria with PICO components, the review strengthens causal inferences, highlights research gaps (e.g., limited DNA repair biomarker data), and provides a template for future studies to standardize protocols in exercise and redox biology research. A comprehensive list of inclusion and exclusion studies is detailed in Supplementary Table S4: Included and Excluded Studies.

2.5. Data Extraction

Following the full-text review, comprehensive data extraction was performed for each included study. Data extraction was conducted independently by two reviewers using Excel spreadsheets, and the information was subsequently verified for accuracy. Study-specific details were also extracted, country of study, including participant characteristics, study design, and data of MLT intervention (route of administration, dosage, and duration of treatment). Biochemical analyses, reported effects, overall study outcomes, and the principal biological conclusions were documented [73,76].

Additionally, special emphasis was placed on identifying and describing the biological pathways implicated in the observed effects. The analysis focused on oxidative stress pathways, as evidenced by the evaluation of classical markers such as malondialdehyde (MDA), advanced oxidation protein products (AOPP), and protein carbonyls, as well as antioxidant defense mechanisms involving enzymes like glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). The studies also explored pathways related to DNA damage and repair, including direct assessment of DNA strand breaks in lymphocytes (comet assay) and indirect markers such as 8-OHdG and protein carbonyls. Furthermore, the modulation of inflammatory and metabolic responses was considered, with measurements of creatinine, uric acid, and hepatic enzymes (ASAT, ALAT, GGT), highlighting the interplay between oxidative stress, cellular damage, and antioxidant protection during and after exercise, particularly in the context of MLT supplementation [38,40].

For all information of the included articles, see Supplementary Table S5: Overview of Included Studies, in Supplementary Materials. After reviewing the references for the articles included in the review, no other articles were considered for additional inclusion because they did not meet our selection criteria.

2.6. Eligibility for Synthesis

To determine eligibility for synthesis, the intervention characteristics and outcome measures of each included study were extracted and tabulated in structured Excel spreadsheets. These tables allowed for detailed comparison of study design elements, biomarker types, dosage and duration of MLT supplementation, and outcome assessments (Supplementary Table S4: Comprehensive Overview of Included Studies). Prior to synthesis, data were prepared by aligning the biomarkers into comparable domains (e.g., oxidative stress, DNA damage, antioxidant activity), and concentration changes before and after the intervention were analyzed relative to the control groups. Visual representation of the relationships between biomarkers and outcomes was also developed through schematic figures to illustrate consistency or divergence among studies. Given the heterogeneity in study designs, intervention protocols, and outcome measures, a narrative synthesis approach was adopted. No meta-analysis was performed due to insufficient standardization across trials. Likewise, no subgroup or sensitivity analyses were conducted. The results were synthesized based on direction and magnitude of biomarker changes and their agreement across studies.

2.7. Risk of Bias

Since none of the included studies reported a self-assessment of risk of bias, we conducted an independent evaluation using the Risk of Bias 2 (RoB 2) tool developed by the Cochrane Collaboration, which is widely recognized as the methodological gold standard for assessing risk of bias in randomized controlled trials (RCTs). The RoB 2 tool evaluates five distinct domains of potential bias: the randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result. For each included study, two reviewers independently applied the structured signaling questions provided by RoB 2 to each relevant domain, ensuring a comprehensive and systematic assessment. Any discrepancies between reviewers were resolved through discussion, and when consensus could not be reached, a third reviewer was consulted. The risk of bias was categorized as low, some concerns, or high for each outcome, in accordance with the RoB 2 guidance. This rigorous approach, including outcome-specific assessments and transparent documentation of judgments, enhances the reliability and reproducibility of our findings and aligns with current best practices as outlined in the Cochrane Handbook for Systematic Reviews of Interventions and the PRISMA 2020 guidelines. Details in Supplementary Table S6. Risk of Bias Assessment.

3. Results

3.1. Literature Search

Study selection followed PRISMA guidelines. A total of 65 records were identified, with 3 articles excluded early due to accessibility or formatting issues. During title and abstract screening, 27 records were excluded: 24 reviews, 1 review correction, 1 short communication, and 1 book chapter. In the full-text eligibility assessment, 29 reports were excluded due to incompatibilities with the PICOS criteria: 23 for inappropriate population (P), 5 for unsuitable intervention (I), and 1 for inadequate outcome (O). For all information on the articles, see Supplementary Table S5: Overview of Included Studies.

3.2. Selection Criteria Studies

A total of six articles were included in the final synthesis based on predefined eligibility criteria (Figure 2). The included studies were published between 2017 and 2023 and exclusively involved human participants. Selection was guided by the PICOS framework, focusing on randomized or controlled clinical trials that evaluated the effects of exogenous MLT supplementation in healthy, physically active adults, with outcomes related to oxidative stress, DNA damage, or DNA repair. All studies met the criteria for methodological rigor and relevance to the review’s objectives. Detailed characteristics of the included studies are presented in

Table 2. Description of the studies included in this review. Summary of major randomized, double-blind clinical trials evaluating the effects of MLT supplementation (MLT) on biochemical biomarkers, oxidative stress (ROS), and DNA repair markers in male athletes. The table shows the study design, sample, intervention (dose, time, and duration), main biochemical biomarkers analyzed, markers of oxidation/oxidative stress, and direct and indirect markers of DNA repair. Arrows indicate changes in parameters: ↑ (increase), ↓ (decrease), ∅ (no change), ↖/↘ (slight increase/decrease), ― (not evaluated). Abbreviations: DM: Direct marker; IM: Indirect marker; MAS: Maximal Aerobic Speed; CK: Creatine Kinase; LDH: Lactate Dehydrogenase; AST: Aspartate Aminotransferase; ALT: Alanine Aminotransferase; GGT: Gamma-Glutamyl Transferase; FA: Alkaline Phosphatase; TG: Triglycerides; WBC: White Blood Cells; NE: Neutrophils; LY: Lymphocytes; MO: Monocytes; SOD: Superoxide Dismutase; GPx: Glutathione Peroxidase; GR: Glutathione Reductase; TAC: Total Antioxidant Capacity; ORAC: Oxygen Radical Absorbance Capacity; LPO: Lipid Peroxidation; NOx: Nitric Oxide Metabolites; AOPP: Advanced Oxidation Protein Products; GSH: Reduced Glutathione; GSSG: Oxidized Glutathione; PLA: Placebo; RAST: Running-based Anaerobic Sprint Test; us-CRP: Ultra-sensitive C-reactive Protein.

DNA Repair Biomarkers	ROS Oxidation Biomarkers	Biochemical Biomarkers	Intervention	Study Design	Number of Samples	Studies (Author- Year)
Indirect markers: Hcy ↓, us-CRP ↓, CK ↓, LDH ↓, AST ↓ Direct markers:―	MDA ↓, WBC ↓, NE ↓, LY ↓, La ∅, GL ∅	CK, LDH, AST, Hcy, us-CRP, Glc, La, WBC, NE, LY, MO	10 mg after evening exercise (10 pm), single dose	Double-blind, randomized, crossover, RAST test (Running-Based Anaerobic Sprint Test)	14 males, teens, (14.5 ± 0.5 yrs)	Cheikh et al. [9]
Indirect markers: Hcy ↓, us-CRP ↓, CK ↓, LDH ↓, AST ↓ Direct markers: ―	ASAT ↓, ALAT ↓, GGT ↓, CREA ↓, WBC ∅, NE ∅, LY ∅, La ∅, GL ∅	ASAT, ALAT, GGT, CREA, TC, HDL, LDL, TG, GL, La, WBC, NE, LY, MO	6 mg MLT, 30 min before exercise (17:00 h ± 30 min), single dose	Randomized double-blind, placebo-controlled (running exercise test (RET) at 100% of their MAS)	12 males soccer players (17.54 ± 0.78 yrs)	Farjallah et al. [45]
Direct markers: DNA damage in lymphocytes assessed by comet assay	TAC ↑ GPx ↑ SOD ∅	MLT, Glc, CREA, UA, TC, TG, TBIL, TP, TRF, ALB	20 mg/day of MLT, taken before exercise, 2-week	Double-blind, randomized, placebo; HIIT (High Intensity Interval Training) strength training	14 male athletes (20–37 years old)	Ortiz-Franco et al. [53]
Indirect markers: CK ↓, ASAT ↓, ALAT ↓ Direct markers: ―	AOPP ↓, GPx ↑, GR ↓, UA ∅, TBIL ↓, WBC ↓, NE ↓, LY ↓	CK, LDH, UA, TBIL	5 mg of MLT orally, taken daily at 7 pm after training, 6-day	Randomized double-blind, placebo-controlled	20 male soccer players (18.81 ± 1.3 yrs)	Farjallah et al. [7]
Indirect markers: CK ↓, LDH ↓ Direct markers: ―	MDA ↑, SOD ↓, GPx ↓, UA ∅, TBIL ∅	CK, LDH, AST, ALT, CREA, Urea, Glc, WBC, NE, LY, MO	6 mg of MLT, taken 30 min pre-exercise (17 h ± 30 min), single dose	Double-blind, randomized, crossover; intensive soccer training	13 male, soccer players (17.5 ± 0.8 yrs)	Farjallah et al. [66]
Indirect markers: GSH ↓, GSSG ↓, GSSG/GSH ↑, GPx ↑, GRd ↑, GPx/GR ↑, CK ↓, LDH ↓, TC ↓, CREA ↓ Direct markers: ―	ORAC ↑, LPO ↓, NOx ↓, AOPP ↖, GPx ↑, GRd ↘	CK, LDH, CREA, TC, TG, Glc, Urea, UA, AST, ALT, WBC, NE, LY, MO	100 mg/day of MLT, oral administration, 30–60 min before bed, 4 weeks	Double-blind, randomized, placebo-controlled; resistance training.	24 male young adults	Leonardo-Mendonça et al. [8]

: Description of the studies included in this review.

3.3. Study Characteristics

Tunisia is the predominant geographical origin of the individuals (66.67%) included in the studies in this literature review. In addition, a European contribution to the data set analyzed was provided by two studies conducted in Granada, Spain. The human study participants were 100% male. They ranged in age from adolescence to adulthood, with an age range of 14–37 years. The majority (66.67%) were young people under 20 years of age. Regarding the number of subjects, studies ranged from 12 to 24 subjects. The review of the literature reveals a categorical preponderance for randomized, double-blind, placebo-controlled trials (RCTs) in human research. Within this robust category, there is a notable frequency of crossover designs, in which participants receive both the intervention and placebo sequentially. This allows effects to be compared within the same individual and potentially reduces interindividual variability. Details in Supplementary Table S5. Comprehensive Overview of Included Studies.

The literature analyzed is consistent in demonstrating the use of MLT—administered orally compared to a control (placebo or no supplementation)—as an intervention in studies with human participants who were exposed to intense physical exercise protocols. The interventions can be categorized as follows: A single dose of MLT (50%), both before and after exercise, and continuous MLT supplementation, ranging from six days to two weeks. Studies consistently demonstrate that exogenous MLT administration exerts beneficial effects in attenuating exercise-induced oxidative stress and cellular damage in athletes. We differentiated between single-dose and continuous supplementation protocols to enhance the comparability of results across studies.

Cheikh et al. [9] (Tunisia), observed that a single 10 mg dose of MLT administered—after physical exercise—to adolescent volleyball players significantly (p < 0.01) reduced markers of muscle damage (CK, LDH, AST), inflammation (p < 0.001) (WBC, NE, MO), and Lipid peroxidation markers (p < 0.05) (MDA), along with improvements in physical performance metrics and reduced perceived exertion. Farjallah et al. conducted Two separate studies [7,45,66]. In the first study, a single 6 mg dose of MLT administered 30 min before exercise led to significant reductions (p < 0.05) in oxidative stress markers such as malondialdehyde (MDA), along with favorable modulation of antioxidant enzymes like glutathione peroxidase (GPx) and glutathione reductase (GR), without negatively impacting athletic performance. In a similar study, using the same 6 mg dose administered prior to exercise, Post hoc analysis showed a significant decrease in SOD level following the running exercise test in participants with melanin ingestion (p < 0.05, d = 0.74), further supporting MLT’s antioxidant and cytoprotective potential under repeated physical stress. Across the three studies, acute MLT supplementation consistently reduced markers of oxidative stress, muscle damage, and inflammation, while preserving or enhancing physical performance under exercise-induced stress.

Leonardo-Mendonça et al. [8] administered 100 mg/day of MLT for 4 weeks to endurance-trained athletes and regarding the plasma LPO, AOPP and NOx levels, observed that MLT more efficiently reduced LPO (p < 0.001), AOPP (p < 0.001), and NOx (p < 0.01) levels compared with the placebo. Additional benefits included improvements in renal and hepatic biochemical parameters, blood lipids, and hematological indices. Additionally, Ortiz-Franco et al. [53] highlighted the significant increase in the antioxidant system in the MLT-treated athletes through a significant increase in total antioxidant capacity levels and GPx activity in comparison with the placebo group. In DNA damage, expressed as percentage of tail intensity and determined by comet assay measured, the MLT group presented significantly less (p < 0.05) DNA damage (37.55 ± 19.68%) than placebo group athletes (58.50 ± 18.98%) after intervention. Reports on the correlation coefficients predicting DNA damage based on the antioxidant status in MLTe group revealed negative associations were found for DNA damage and antioxidant status in MG after intervention (r = −0.679; p = 0.047) where less DNA damage was associated with better antioxidant status. Farjallah et al. [7] conducted a continuous 5 mg daily dose over six days during repeated sprint. Compared to placebo, MLT intake decreased resting oxidative stress markers (i.e., advanced oxidation protein products), leukocytosis (i.e., white blood cells (WBC), neutrophils (NE) and biomarkers of cellular damage (i.e., creatine kinase (CK)). For AOPP, after the training camp, levels increased only in the placebo group (p < 0.01, d = 2.67) and its level became higher than that of the MLT group (p < 0.01, d = 2.29). GPx activity after exercise increased only in the MLT group (p < 0.01, d = 1.54). Across all three studies, continuous MLT supplementation consistently demonstrated protective effects against exercise-induced oxidative stress, cellular damage, and inflammation, while also supporting antioxidant capacity and improving hepatic and hematological parameters.

3.4. Results of Syntheses

Despite variations in duration and intensity, the exercise protocols employed across the reviewed studies are considered viable for inducing disturb antioxidant status of competitive athletes. This is substantiated by consistent changes in biochemical markers.

Regarding biochemical biomarkers, there was considerable variability among the included studies, with no single biomarker evaluated consistently across all trials. However, several markers were frequently assessed, particularly creatine kinase (CK) and lactate dehydrogenase (LDH), which were measured in Farjallah et al. [7,66], Cheikh et al. [9], and Farjallah et al. [7]. Additionally, markers such as creatinine and urea were commonly reported by Farjallah et al. [66], Ortiz-Franco et al. [53], Leonardo-Mendonça et al. [8], and Farjallah et al. [7]. Other biomarkers and various liver enzymes (CK, LDH, WBC, NE and LY), were also frequently evaluated but with less consistency across studies [7,8,9,66]. These findings highlight a general focus on muscle damage, renal function, and metabolic stress parameters in investigations of MLT supplementation during exercise.

There was no single oxidative stress biomarker evaluated across all studies (Figure 3). Regarding oxidative stress biomarkers, superoxide dismutase (SOD) was assessed in three studies [7,45,53,66]. Glutathione peroxidase (GPx) and glutathione reductase (GR/GRd) were each evaluated in four studies [7,8,45,53,66]. Advanced oxidation protein products (AOPP) were also analyzed in four studies [7,8,45,53,66]. Malondialdehyde (MDA) was measured in three studies [7,9,45]. Details in Figure 3. Only Ortiz et al. [53] used the comet assay (lymphocytes) as a direct biomarker of DNA damage. Other authors did not report this evaluation.

This methodological heterogeneity hinders direct comparisons between findings and limits the ability to draw definitive conclusions regarding the effectiveness of MLT in mitigating oxidative stress and protecting DNA integrity. Moreover, the figure highlights that some studies—particularly those by Leonardo-Mendonça et al. and Ortiz-Franco et al. [8,53]—assessed a broader range of biomarkers, reflecting greater methodological rigor in characterizing redox status. Thus, the circle plot serves as a valuable visual tool for synthesizing the biomarker data reported across the included studies and substantiates the authors’ call for greater standardization in future research protocols addressing this topic [77].

3.5. Quality Assessment

3.5.1. Methodological Approach

A comprehensive assessment of methodological quality was conducted for all included studies using the revised Cochrane Risk of Bias tool for randomized trials (RoB 2) [78]. This standardized framework evaluates potential sources of bias across five critical domains that can influence the validity of trial results. Two independent reviewers performed the assessments, with disagreements resolved through consensus discussion or consultation with a third reviewer. For full information on the conceptual risk of bias analysis, see: Supplementary Table S6: Risk of Bias Assessment.

The assessment focused on the intention-to-treat effect, as documented in Figure 4, and evaluated each study across the following domains:

• Bias arising from the randomization process
• Bias due to deviations from intended interventions
• Bias due to missing outcome data
• Bias in measurement of the outcome
• Bias in selecting the reported result

Each domain received a judgment of “low risk,” “some concerns,” or “high risk” of bias according to responses to specific signaling questions and established algorithms [76]. The overall risk of bias judgment for each study followed the principle that the study’s overall risk corresponds to the highest risk of bias assigned to any single domain.

3.5.2. Results of Risk of Bias Assessment

Our analysis demonstrated a generally favorable methodological quality across the included studies (Figure 4). All studies (100%) were assessed as having low risk of bias in the domains of measurement of the outcome, missing outcome data, deviations from intended interventions, and randomization process. However, approximately 50% of studies presented “some concerns” regarding selection of the reported result and, consequently, in the overall bias assessment.

The most common methodological limitations identified were:

• Lack of pre-registered protocols or statistical analysis plans (Domain 5)
• Incomplete reporting of randomization procedures in some studies, although baseline characteristics were generally balanced between groups
• Insufficient detail regarding allocation concealment methods

Despite these limitations, the double-blind design employed in all studies and the use of objective laboratory measurements for outcomes contributed significantly to reducing potential bias.

Table 3. Summary of Risk of Bias (RoB 2) Domains in the Included Studies *.

Global Risk	Bias in Selecting Results	Bias in Measurement	Bias in Missing Data	Bias in Deviations	Bias in Randomization	Key Outcomes	Intervention	Authors (Year)
Low	Low	Low	Low	Low	Low	Muscle damage, oxidative stress	MLT 10 mg post-exercise	Cheikh et al. [9]
Low	Low	Low	Low	Low	Low	Hepatic/ rhenaic markers	MLT 6 mg pre- exercise maximum	Farjallah et al. [45]
Low	Low	Low	Low	Low	Low	Antioxidant capacity, DNA damage	MLT 20 mg/day + HIIT	Ortiz-Franco et al. [53]
Some concerns	Some concerns	Low	Low	Low	Low	Oxidative stress, performance	MLT 5 mg during training	Farjallah et al. [7]
Some concerns	Some concerns	Low	Low	Low	Low	Oxidative stress, muscle damage	MLT 6 mg pre-race	Farjallah et al. [66]
Some concerns	Some concerns	Low	Low	Low	Low	Status redox, dano muscular	MLT 100 mg/day before bed	Leonardo-Mendonça et al. [8]

* Summary of the risk of bias (RoB 2) assessment for each included study, showing judgments for each domain and the overall risk of bias. Judgments are categorized as “Low risk,” “Some concerns,” or “High risk” according to the Cochrane RoB 2 tool. For full information on the conceptual risk of bias analysis, see Supplementary Table S6: Risk of Bias Assessment, in Supplementary Materials.

presents a summary of the risk of bias (RoB 2) assessment for each included study, detailing judgments for each domain-randomization, deviations from intended interventions, missing data, measurement of outcomes, and selection of reported results-as well as the overall risk of bias. The evaluations are categorized as “Low risk,” “Some concerns,” or “High risk,” in accordance with the Cochrane RoB 2 tool. This structured presentation enables transparent comparison of methodological rigor across studies and highlights domains where bias may influence interpretation of results.

3.5.3. Implications for Evidence Interpretation

The quality assessment findings have important implications for interpreting the evidence synthesized in this review. The overall methodological rigor observed supports confidence in the reported outcomes regarding MLT’s effects on exercise-induced oxidative stress markers. However, the “some concerns” rating for selection of reported results in approximately half of the studies suggests caution when interpreting these findings, particularly regarding the potential for selective outcome reporting [76,79,80,81].

The heterogeneity in measurement methods and biomarkers used across studies (as illustrated in Figure 3) further complicates the interpretation of pooled results. Future research would benefit from standardization of core outcome sets for exercise-induced oxidative stress studies and increased adherence to trial registration and complete reporting as advocated by the CONSORT guidelines. The detailed assessment for individual studies is available in Supplementary Table S6: Risk of Bias Assessment, providing transparency regarding the specific strengths and limitations of each included trial [76,79,80].

3.6. Convergent Synthesis of the Risk of Bias

The convergent synthesis of risk of bias across the included studies highlights several methodological strengths and persistent limitations that influence the interpretation of MLT’s effects on exercise-induced oxidative stress and DNA damage. The circle plot (Figure 3) visually demonstrates the heterogeneity in biomarker selection, with frequent assessment of malondialdehyde (MDA) and glutathione peroxidase (GPx), but only a single study directly measuring DNA damage via comet assay. This lack of biomarker standardization underscores a critical gap in the current literature and complicates direct comparisons and meta-analytic synthesis [76,82,83].

Across the five domains of the revised Cochrane Risk of Bias tool (RoB 2), most studies demonstrated low risk in randomization, deviations from intended interventions, missing outcome data, and measurement of outcomes. These strengths are attributable to the widespread use of double-blind, placebo-controlled designs, objective laboratory endpoints, and high participant retention. However, “some concerns” were consistently identified in the domain of selection of the reported result, primarily due to the absence of pre-registered protocols and insufficient detail regarding statistical analysis plans. This limitation raises the possibility of selective outcome reporting, particularly as studies tended to emphasize positive findings related to antioxidant effects while underreporting null or negative results for DNA repair endpoints [78,79,83].

The methodological heterogeneity is compounded by differences in MLT dosing regimens (ranging from single acute doses to four-week supplementation), exercise protocols, and participant characteristics. Such variability, combined with small sample sizes (n = 12–24), limits the generalizability and reproducibility of findings. Notably, the predominance of male participants and exclusion of individuals with comorbidities may restrict the applicability of results to broader athletic or clinical populations [74,79,82].

Despite these limitations, the internal validity of included studies is supported by rigorous blinding, objective outcome measurement, and complete data reporting. The consistent reduction in oxidative stress markers (e.g., MDA, AOPP) and muscle damage indicators (CK, LDH) across studies strengthens the evidence for MLT’s cytoprotective effects in the context of exercise. However, the paucity of direct DNA damage and repair assessments-highlighted by the use of comet assay in only one trial-prevents definitive conclusions regarding the genoprotective mechanisms of MLT [39,84,85,86].

These findings reinforce the urgent need for consensus on core outcome sets and standardized biomarker panels in exercise-induced oxidative stress research, as advocated by recent methodological frameworks. Future studies should prioritize the inclusion of direct DNA damage and repair markers (e.g., comet assay, 8-OHdG, BER enzyme activity), pre-registration of protocols, and adherence to CONSORT reporting guidelines to enhance transparency, reproducibility, and the robustness of evidence synthesis [72].

3.7. Exclusion

In the context of a systematic review investigating the effects of exogenous MLT administration on DNA repair mechanisms in response to exercise-induced oxidative stress, the careful selection of study populations is paramount for ensuring the internal validity and translational relevance of the findings. The list of excluded articles provided encompasses a diverse array of populations, including patients with chronic diseases such as hemodialysis, chronic obstructive pulmonary disease (COPD), heart failure, and obesity, as well as various animal models subjected to non-exercise-related interventions or possessing underlying pathologies unrelated to the review’s central focus [10,31,72,79,87]. The exclusion of these populations is justified on both methodological and biochemical grounds, as outlined below (Detail in Supplementary Table S3: Comprehensive Exclusion Criteria).

4. Discussion

Exercise-induced oxidative stress is a well-documented phenomenon that can lead to cellular damage, including DNA lesions. MLT, a hormone known for its antioxidant properties, has been proposed as a potential countermeasure [13,54,88,89]. This systematic review evaluates data from six clinical trials that evaluate the effects of exogenous MLT administration on exercise-induced oxidative stress. Results suggest that MLT has a protective effect on modulating oxidative stress biomarkers, with limited but promising evidence regarding DNA protection.

We interpreted the observed variations in biochemical and redox biomarkers following exercise protocols as experimental evidence that the physical interventions effectively induced systemic stress. This physiological response suggests that the exercise regimen created appropriate conditions for a human model to evaluate the effects of MLT supplementation. Given MLT’s established antioxidant and anti-inflammatory properties, its administration in this context allows for the assessment of its potential to mitigate exercise-induced oxidative stress and inflammation [9,38,42,90].

4.1. Classification of Biomarkers as Direct or Indirect

A critical consideration in interpreting the results of exercise-induced oxidative stress studies is the classification of biomarkers as direct or indirect, as this distinction fundamentally influences both mechanistic understanding and clinical translation. Direct biomarkers, such as the comet assay or quantification of specific oxidative DNA lesions (e.g., 8-hydroxy-2′-deoxyguanosine), provide immediate evidence of DNA strand breaks or base modifications, thereby offering a precise assessment of genomic integrity following acute oxidative insults [2,39,58,91].

In the present review, the comet assay was the only direct biomarker employed, demonstrating a significant reduction in DNA damage with MLT supplementation. However, the majority of included studies relied on indirect biomarkers, such as malondialdehyde (MDA) for lipid peroxidation, advanced oxidation protein products (AOPP) for protein oxidation, and the activities of antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GPx) [52,53,92,93]. These indirect markers reflect the downstream consequences of reactive oxygen species (ROS) overproduction or the adaptive capacity of endogenous defense systems, rather than measuring ROS or DNA lesions directly. While indirect biomarkers are valuable for capturing the cumulative oxidative burden and the efficacy of antioxidant interventions, they may not always correlate with the extent of DNA damage or repair. Clinically, this distinction is pivotal: direct biomarkers are essential for evaluating the true genoprotective efficacy of interventions such as MLT, whereas indirect biomarkers provide broader context regarding systemic redox status and tissue injury. Therefore, future studies should prioritize the integration of both direct and indirect biomarkers to achieve a comprehensive and clinically meaningful assessment of oxidative stress and DNA stability in the context of exercise and antioxidant supplementation [4,10,13,31,39,42,45,91,92].

4.2. Physiological Conditions/Baseline

Creatine kinase was examined in all the reviewed studies due to its relevance as a biomarker for muscle stress and damage, especially in response to intense physical activity. While regular exercise does not usually lead to significant elevations in creatine kinase, levels may increase when the intensity exceeds the individual’s typical training load. Additionally, this biomarker is often studied alongside aminotransferases such as glutamic oxaloacetic transaminase (GOT) and aspartate aminotransferase (AST), which may also indicate muscle damage [40]. Serum creatinine was another frequently used marker. As it is produced at a relatively constant rate under normal physiological conditions and could be related to skeletal muscle mass, serum creatinine serves as an important indicator in metabolic evaluations. For example, older adults with sarcopenia usually exhibit lower serum creatinine levels when compared to athletes, highlighting its utility in studies focusing on physical performance and muscle metabolism [86,94,95].

In addition to these biomarkers, glucose was also explored in several studies [96,97]. Glucose plays a central role in energy production, and during aerobic activity, it is primarily disposed of in skeletal muscle. When carbohydrate availability is limited, the liver maintains metabolic homeostasis through the utilization of alternative substrates such as lactate, fatty acids, and amino acids. Although glucose is essential for understanding the metabolic response to exercise, lactate has emerged as a more promising precursor of oxidative metabolism under these conditions [43]. These biomarker responses confirm that the exercise protocols effectively induced systemic stress, supporting their validity as experimental models. This provides a solid basis for evaluating MLT’s potential to modulate oxidative stress and related physiological responses in humans.

4.3. ROS

Among the biomarkers assessed, the most consistent variables across the studies were superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPx), and advanced oxidation protein products (AOPP). These markers were commonly used to evaluate the antioxidant response and oxidative damage triggered by physical activity. MDA and AOPP levels, which are indicative of lipid and protein oxidation, respectively, showed variable changes, often reflecting the intensity of the exercise protocol and the timing of sample collection [7,9,45,66]. In contrast, SOD and GPx, as key endogenous antioxidant enzymes, were frequently reported to increase following MLT supplementation, suggesting an enhancement of the body’s defense mechanisms against reactive oxygen species (ROS) [98]. The repeated use of these specific biomarkers reinforces their relevance and reliability in assessing redox homeostasis and the potential modulatory role of MLT under exercise-induced oxidative stress.

4.4. DNA Damage

Of relevance was the demonstration in one of the studies of a significant reduction in DNA damage (assessed by comet assay) following MLT supplementation in athletes undergoing high-intensity exercise. This genoprotective potential is further supported by quantitative evidence showing that athletes who received MLT exhibited significantly lower DNA damage, compared to the placebo group [53]. Moreover, a moderate negative correlation was observed between DNA damage and antioxidant status in the MLT group after intervention, indicating that higher antioxidant capacity was associated with reduced DNA fragmentation. Although only one of the included studies directly assessed DNA damage, we consider that the remaining studies provide indirect support for these findings through consistent improvements in redox biomarkers and reductions in cellular damage indicators associated with oxidative stress [10,13,31,53].

The reviewed evidence demonstrates that omics methodologies are pivotal in elucidating MLT’s protective mechanisms against exercise-induced DNA damage. Proteomic studies consistently show MLT upregulates DNA repair proteins (e.g., BRCA1, RAD51) and antioxidant enzymes, mitigating oxidative stress in skeletal muscle [3,12]. Metabolomic analyses further correlate MLT supplementation with reduced lipid peroxidation markers and enhanced glutathione recycling, suggesting improved redox homeostasis [49]. However, gaps persist in lipidomic profiling of oxidative byproducts like 4-hydroxynonenal, which could clarify MLT’s role in membrane integrity preservation during intense exercise. While current data emphasize transcriptomic and proteomic adaptations, integrating multi-omics platforms may uncover synergistic pathways linking MLT’s antioxidant properties to epigenetic regulation of DNA repair genes. Future studies should prioritize longitudinal omics designs to map temporal dynamics of MLT’s effects across tissue types and exercise intensities [3,10].

This review suggests that MLT supplementation could be a promising strategy for attenuating oxidative stress and reducing cell damage associated with intense exercise. The potential protective effect of MLT against DNA damage, although based on limited evidence, suggests a new direction for the use of MLT in sports medicine and post-exercise recovery. Nonetheless, the effectiveness of MLT’s genoprotective mechanisms is closely linked to its pharmacodynamic and pharmacokinetic properties.

The wide heterogeneity in supplementation protocols—particularly regarding dosage (ranging from 5 to 100 mg), timing of administration (pre- vs. post-exercise), and duration (single vs. multi-day)—significantly limits direct comparisons and complicates dose–response interpretations across studies. The variability in supplementation protocols and exercise regimens between studies must be interpreted with caution. Consequently, the choice of formulation—immediate vs. prolonged release—directly influences its therapeutic potential by modulating the timing and duration of its action on DNA-protective pathways [99]. MLT is an endogenous metabolite that, when administered exogenously as a dietary supplement, typically exhibits a short biological half-life. This property is particularly evident in immediate-release formulations, which generally achieve peak plasma concentrations within approximately 15 to 20 min post-administration, resulting in short-term therapeutic efficacy [99]. Nevertheless, due to the rapid absorption and clearance of MLT, such formulations often necessitate repeated dosing and higher concentrations to sustain the intended pharmacological effects [100]. In contrast, extended-release or prolonged-release MLT formulations have been developed to more closely mimic the physiological secretion profile of the hormone. Clinical studies have demonstrated that these formulations maintain plasma MLT concentrations above 50 pg/mL for up to 10 h, thereby enhancing therapeutic effectiveness, particularly in the management of insomnia [101,102]. Timing and formulation choice are therefore critical, since ER MLT can maintain protective plasma concentrations overnight or across multi-day recovery phases, potentially enhancing resistance to exercise-induced oxidative stress, particularly following late or nocturnal training sessions [103]. Nonetheless, further research is needed to establish optimal dosing strategies tailored to exercise type and intensity.

4.5. Biological Pathways

MLT can exert genoprotective effects through multiple mechanisms and biological pathways, some of which may explain the results observed in the studies. Based on the evidence reviewed, we propose that the following findings support a potential protective role of MLT in DNA repair in the context of exercise-induced oxidative stress. It is important to highlight that these mechanisms can interact and complement each other.

• Direct free radical scavenging: MLT functions as a direct scavenger of reactive species, neutralizing radicals such as hydroxyl (-OH) and peroxynitrite (ONOO⁻), both of which are highly damaging to DNA. Studies, such as that by Farjallah et al. [66], have shown that MLT supplementation (6 mg/day) significantly reduces lipid peroxidation, measured by malondialdehyde (MDA), in athletes after exhaustive sprints (2.22 ± 1.30 vs. 2.89 ± 0.77 µmol/L in the placebo group; p < 0.05). This reduction in oxidative stress decreases the likelihood of DNA damage, as unneutralized free radicals can cause double-strand breaks and modifications of nitrogenous bases, such as the formation of 8-OHdG.
• Increased activity of endogenous antioxidant enzymes: MLT can modulate the expression and activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), reinforcing cellular defense against oxidative stress [27,54,98]. The study you mentioned observed an increase in GPx activity with MLT supplementation, which may contribute to DNA protection.
• Modulation of Inflammation: MLT demonstrates significant anti-inflammatory effects that complement its antioxidant mechanisms in protecting DNA during intense exercise. Strenuous physical activity induces inflammatory responses characterized by elevated leukocyte counts (WBC) and neutrophil (NE) activation, which exacerbate oxidative stress and genomic damage [7,9,45,66]. Experimental data reveal that MLT administration suppresses post-exercise leukocytosis, with placebo groups exhibiting markedly higher WBC levels (p < 0.001). This hormone also reduces neutrophil infiltration into tissues (p < 0.001) and lowers concentrations of systemic inflammatory markers such as us-CRP [7,9,45,66].
• At the molecular level, MLT inhibits the production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), through suppression of nuclear factor-kappa B (NF-κB) signaling pathways [104]. This mechanism is particularly relevant during high-intensity exercise, which typically increases circulating levels of these inflammatory mediators. By attenuating NF-κB activation, MLT prevents the overexpression of cytokines responsible for exercise-induced muscle damage and subsequent oxidative stress. The combined action of direct free radical neutralization and indirect inflammatory pathway modulation enhances genomic stability under physiological stress conditions [27,54,98].
• Mitochondria protection: Mitochondria is an important source of ROS during exercise. MLT can protect mitochondria from oxidative damage, preserve their function and reduce the production of free radicals in this organelle, contributing to the protection of nuclear and mitochondrial DNA [6] (Figure 5). MLT improves mitochondrial function by reducing ROS production and enhancing ATP synthesis. Farjallah et al. [7,45,66] demonstrated that MLT increased mitochondrial membrane fluidity in hepatocytes (p < 0.05), likely via activation of sirtuin-1 (SIRT1), which regulates mitochondrial biogenesis and oxidative phosphorylation.

4.6. Strengths and Limitations

A key strength of this systematic review lies in its focused inclusion criteria, which limited the analysis to studies involving healthy human participants and exercise-induced oxidative stress, thereby enhancing the translational relevance of the findings. Additionally, the review followed PRISMA guidelines and employed a systematic search strategy, ensuring methodological rigor and minimizing the risk of bias in study selection and data synthesis [72,79].

The heterogeneity observed among the included studies, in terms of participant demographics, exercise regimens, and MLT dosing protocols, poses challenges in drawing definitive conclusions. Future research should aim for standardized methodologies to facilitate more accurate comparisons and meta-analyses. Athletes, due to their chronic exposure to high-intensity physical activity, may already present an adaptive baseline characterized by enhanced antioxidant defenses and DNA repair capacity. Consequently, the magnitude of exercise-induced DNA damage in this group may be lower compared to untrained individuals, potentially attenuating the observable effects of MLT supplementation. This attenuated response may also be less prominent in studies involving continuous dosing protocols.

Risk of Bias and Methodological Limitations

The risk of bias assessment conducted using the RoB 2 tool revealed that, overall, the methodological quality of the included studies was moderate. Most domains—such as the randomization process, deviations from intended interventions, missing outcome data, and measurement of the outcome—were consistently rated as low risk of bias across all studies. However, two domains presented some concerns, namely the selection of the reported result and the overall bias, with approximately 50% of the studies rated in this intermediate category. These concerns may reflect insufficient reporting of pre-specified outcomes or lack of transparency in selective reporting. Despite these limitations, no study was judged to have a high risk of bias in any domain, suggesting that the evidence base is reasonably reliable, though future studies should improve methodological reporting and pre-registration practices to enhance transparency and reproducibility [78,79,83,105].

Limitations of the Review Process. A key limitation of this review is the use of a single database—PubMed—for literature retrieval. Although PubMed is a leading source in biomedical and health sciences, restricting the search to this platform may have limited the comprehensiveness of the review and excluded relevant studies available in other databases. The number of studies retrieved was relatively low, suggesting that a broader search strategy could improve coverage in future reviews.

4.7. Future Perspectives

As a future perspective, it is essential to encourage the use of more sensitive and specific methodologies to directly assess DNA damage in exercise-related studies. Although current investigations often rely on indirect markers of oxidative stress, direct evaluation of DNA integrity would provide more robust and mechanistic insights. The comet assay, recognized as a gold-standard method for detecting DNA strand breaks at the single-cell level, offers an accessible and powerful tool for this purpose [92]. Additionally, the measurement of circulating cell-free DNA (cfDNA) in blood, a candidate biomarker initially explored in oncology, could serve as a non-invasive indicator of cellular damage resulting from intense physical activity, given that DNA fragment patterns differ between apoptosis and necrosis [106]. Furthermore, advanced techniques such as ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) are emerging as gold-standard approaches for the precise quantification of DNA modifications, including oxidative and epigenetic alterations [46]. Integrating these methodologies into future exercise and supplementation studies could significantly enhance the understanding of the biological effects of MLT on DNA stability.

Current evidence in physical activity research is predominantly derived from studies involving exclusively male cohorts, primarily adolescents and young adults. This demographic homogeneity limits the generalizability of findings across sexes and age groups. The underrepresentation of female participants may stem from multifactorial challenges, including the hormonal variability inherent to female physiology and the potential confounding effects of oral contraceptive use, which complicate experimental standardization [107]. Moreover, males tend to exhibit higher voluntary participation rates in exercise-related trials, contributing to enrollment bias [105]. Similarly, older adults are rarely included in such studies, despite age-related differences in oxidative stress responses, hormonal regulation, and MLT metabolism. These limitations underscore critical knowledge gaps regarding sex- and age-specific physiological responses to physical exertion and MLT supplementation. Future studies must prioritize the inclusion of female and older populations to elucidate the impact of biological dimorphism and age-associated changes on exercise-induced oxidative stress and DNA repair mechanisms. Addressing these methodological shortcomings will enhance the translational applicability of findings and support the development of tailored strategies in sports medicine and public health.

To optimize MLT supplementation strategies, future studies should systematically compare doses, administration times, and formulations across diverse athlete populations and exercise modalities while adhering to CONSORT guidelines to ensure rigorous reporting of randomization, blinding, and protocol registration. Long-term investigations are critical to evaluate chronic use safety and efficacy, particularly given the heterogeneity in current oxidative stress biomarker reporting and methodological limitations in existing trials. Integrating these elements will enhance reproducibility and clarify dose–response relationships, addressing gaps identified in the present synthesis.

5. Conclusions

Current evidence suggests that exogenous MLT may protect against exercise-induced oxidative stress and potentially attenuate DNA damage. However, most of the included studies assessed only indirect oxidative stress markers, with limited direct assessment of DNA damage or repair. To confirm these effects and better understand the underlying mechanisms, future clinical trials should incorporate validated biomarkers of DNA integrity and repair (e.g., comet assay, enzyme activity assays such as OGG1, APE1), as well as advanced genomic stability tests.