Vitamin C, also known as ascorbic acid, is a water-soluble vitamin that plays a crucial role in the maintenance of overall health. Vitamin C has been extensively studied for its potential health benefits, including managing common cold symptoms [9,10], enhancement of immune function [11,12], and aiding in collagen synthesis [13,14]. Additionally, it may have positive effects on cardiovascular health [15-17] and improve iron absorption [18,19]. It is an essential nutrient as the human body cannot produce or store it in significant amounts, necessitating regular dietary intake. One of the primary functions of vitamin C is its potent antioxidant activity. As an antioxidant, it protects cells from the damage caused by free radicals. Free radicals are highly reactive and can cause oxidative stress, which has been linked to various chronic diseases, such as cardiovascular diseases, certain cancers, and age-related macular degeneration [20].

Ascorbate is the reduced active form of vitamin C. It acts as a powerful antioxidant by readily donating electrons to neutralize free radicals and ROS. By donating an electron, ascorbate stabilizes and neutralizes these harmful molecules, thereby protecting cells and tissues from oxidative damage. This process converts ascorbate into its oxidized form known as dehydroascorbate (Figure 1). Dehydroascorbate is the oxidized form of ascorbate. Dehydroascorbate can be transported across cell membranes, thereby allowing it to move between different cellular compartments. Once inside the cell, dehydroascorbate is enzymatically reduced to ascorbate, regenerating the active form of vitamin C. The interconversion between ascorbate and dehydroascorbate is an essential part of the antioxidant defense system. Ascorbate serves as a primary antioxidant, directly neutralizing free radicals, whereas dehydroascorbate acts as a mediator. The balance between ascorbate and dehydroascorbate is crucial for the overall antioxidant function of vitamin C. Vitamin C plays a vital role in recycling other antioxidants such as vitamin E. Vitamin E, a fat-soluble antioxidant, protects cell membranes from oxidative damage. After vitamin E neutralizes the free radicals, it is oxidized. Vitamin C can then interact with the oxidized form of vitamin E and restore its antioxidant function, allowing it to continue protecting cells [21]. Excessive consumption of vitamin C, that is, exceeding 1 g/day, may lead to adverse effects, such as gastrointestinal disturbances, metabolic acidosis, kidney stones, fatigue, and infertility. However, these effects are associated with the intake of vitamin C exceeding 1 g over several weeks [22,23]. However, supplementation with daily doses of 0.2 g or more but less than 1 g of vitamin C successfully reduces oxidative stress [24,25].

Vitamin C supplements are widely utilized by athletes because of their potential to enhance muscle recovery after intense exercise, which is attributed to their antioxidative effects [26,27]. Various studies have been conducted on vitamin C supplementation and exercise; however, the results have been inconsistent. In a study conducted by Thompson et al. on healthy males, a 2-week supplementation of vitamin C demonstrated beneficial effects on muscle soreness and function, as well as a significant reduction in plasma interleukin-6 (IL-6) levels following 90 minutes of prolonged intermittent shuttle-running test, compared to the placebo group [28]. This finding suggests the effectiveness of vitamin C supplementation in aiding post-exercise recovery. Bryer and Goldfarb observed that a 2-week vitamin C supplementation regimen in healthy males, followed by 4 days of eccentric exercise increased the oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, thereby preventing exercise-induced oxidative stress compared to the placebo group [29]. Evans et al. reported that 28-day vitamin C supplementation increased the peak muscular pushing force and reduced exercise-induced oxidative stress [30]. Other studies have reported the positive effects of vitamin C supplementation on the reduction of exercise-induced oxidative stress [31-33]; however, some studies have also shown that vitamin C supplementation has no impact or even negative effects on exercise performance and training adaptations. Gomez-Cabrera et al. reported a 9.3% reduction in maximal oxygen uptake with the consumption of 1,000 mg of vitamin C for 8 weeks [34]. Braakhuis et al. reported that 3 weeks of vitamin C supplementation during training decreases mean running speed in a 5 km running time in female runners [35]. In contrast, Roberts et al. reported that daily vitamin C supplementation (1000 mg/day) during 4 weeks of interval training did not interfere with training adaptation [36]. Several studies have investigated the effects of combining vitamin C with other antioxidants. Jourkesh et al. found that 3 weeks of 400 mg of vitamin E along with 1000 mg of vitamin C was effective in improving aerobic power [37]. Male athletes supplemented with an antioxidant complex, including vitamin C, exhibited higher training adherence [38,39]. However, several studies have reported that vitamin C and/or E supplementation does not improve exercise performance [40-43]. This discrepancy may be due to differences in participant status, exercise protocol, an-tioxidant dosage, form of intake, duration of intake, timing of intake, and methods for assessing oxidative stress [44]. In a review of vitamin C, vitamin E, and exercise adaptation, Nikolaidis et al. underscored the difficulty in determining the impact of vitamins C and E intake on chronic exercise adaptation and emphasized the need for future research involving appropriate exercise stimulus selection and reliable oxidative-reduction biomarker analyses at various post-exercise time points. Furthermore, they highlighted the need for studies evaluating antioxidant supplementation in welltrained individuals and employing effective and trustworthy testing protocols [45].

Glutathione, a tripeptide composed of glutamate, cysteine, and glycine, is the cornerstone of the antioxidant defense system. Its ability to counteract oxidative stress stems from its dual role as a direct scavenger of ROS and a critical regulator of cellular redox balance. Glutathione protects antioxidants by donating electrons to neutralize harmful free radicals, thus preventing oxidative damage to cellular components, such as proteins, lipids, and DNA [46]. Glutathione operates in an intricate cycle with enzymatic partners such as glutathione peroxidase and glutathione reductase. These enzymes facilitate the conversion of glutathione into its oxidized form (GSSG) and subsequently regenerate the reduced form (GSH) from GSSG. This cycle ensures a continuous supply of reduced glutathione, amplifying its capacity to combat oxidative stress and maintain cellular health [7]. Glutathione has a broad influence on cellular functions. It plays a pivotal role in modulating signaling pathways, gene expression, and immune responses, thereby influencing cellular adaptation to stress and maintaining overall homeostasis [47]. The recently reported roles of GSH in defending cells against oxidative stress and its involvement in protein modification and cellular signaling highlight the importance of the maintenance of intracellular GSH for cellular function and viability [48,49]. The decline in glutathione levels in the plasma and tissues has been linked to aging and disease. Glutathione depletion is particularly linked to neurodegenerative disorders, pulmonary diseases, immune disorders, cardiovascular diseases, and chronic geriatric conditions [50].

With the established association between glutathione levels and aging and disease, various studies have been conducted to explore methods for elevating glutathione levels. The effectiveness of oral glutathione in increasing intracellular glutathione levels has been much debated [51,52]. Some studies have indicated that acute or chronic oral glutathione does not significantly affect glutathione levels or oxidative stress. Witsch et al. found that oral supplementation with 3 g/day GSH did not alter blood GSH levels, while Allen et al. found no change in blood GSH levels or oxidative stress with 500 mg of glutathione supplementation twice daily for 4 weeks [53,54]. However, the evidence is conflicting. Richie et al. conducted a study involving 54 healthy adult males and reported that long-term oral glutathione (GSH) supplementation led to an increase in GSH levels in both blood and red blood cells and a reduction in oxidative stress [55]. Similarly, Park et al. found that, while oral glutathione supplementation did not induce changes in GSH levels in the plasma and cellular compartments, a significant increase was observed in the plasma protein-bound fraction [56]. Kovacs-Nolan et al. investigated the transportability of glutathione in intestinal epithelial cells in vitro. They showed that intestinal epithelial cells can transport glutathione. This challenges the previous notion and suggests that oral glutathione may not undergo significant degradation during digestion. Their findings contradict the theory that oral glutathione breaks down during digestion because of its inability to pass through these cells [57]. Owing to conflicting data regarding oral glutathione supplementation, attention has been directed toward the liposomal [58] or sublingual [59] forms of glutathione. The supplementation of NAC (N-acetylcysteine), which is essential for glutathione synthesis, has been suggested to increase glutathione levels. NAC has shown promise as a supplement for elevating glutathione levels and potentially alleviating certain issues associated with oxidative stress [60,61]. However, research on this topic is inconclusive, and some studies have reported minimal impact of NAC supplementation [62-65]. There is no officially established daily intake of glutathione; however, when used as an ingredient in pharmaceuticals or health functional foods, daily doses of approximately 50-500 mg are commonly used. Several clinical studies have indicated that a daily intake of 250-1000 mg can have significant beneficial effects [58,66]. Furthermore, recent clinical trials administering oral glutathione supplements (250 and 1000 mg/day) for 1, 3, and 6 months demonstrated doseand time-dependent increases in blood glutathione levels. In particular, long-term supplementation with 1000 mg/day for 6 months increased glutathione levels in the plasma, red blood cells, and oral cells, with no reported serious adverse effects [55]. Dilokthornsakul et al. reported minor side effects, such as a feeling of heaviness, reduced skin moisture, and flushing [67], but these effects were not severe. It is worth noting that some glutathione is synthesized in the body, and excessively consumed glutathione is excreted through the urine [68].

During exercise, heightened metabolic demands and increased oxygen consumption can lead to ROS generation in skeletal muscle cells. In the primary defense mechanism, glutathione serves as a key antioxidant by neutralizing ROS and quenching oxidative damage [7]. However, prolonged or intense physical activity can deplete intracellular GSH levels because GSH is oxidized to its disulfide form, glutathione disulfide (GSSG), during ROS detoxification [69,70]. The reduction in GSH availability may contribute to oxidative stress and muscle fatigue, potentially compromising exercise performance and recovery. To address the decrease in GSH levels due to exercise, researchers have investigated the efficacy of GSH supplements in enhancing exercise performance and mitigating oxidative stress. However, only a limited number of studies have been conducted on this topic. Aoi et al. demonstrated that healthy males who engaged in 60 minutes of cycling at 40% of their HRmax intensity after glutathione supplementation exhibited a less pronounced decrease in plasma glutathione levels and suppressed the increase in blood lactate concentration compared with the placebo group. This improved muscular aerobic metabolism during exercise, consequently reducing muscle fatigue [66]. Petrov et al. reported that 24 elite swimmers supplemented with 250 mg of glutathione daily during six weeks of training showed a significant decrease in swim time trials compared with the controls [71]. Owing to negative perceptions surrounding oral glutathione intake, many researchers have focused on NAC supplementation or intravenous injections as alternatives to oral glutathione administration. Supplementation with NAC(1200 mg/day) for 7 days improved muscle fatigue during the graded exercise treadmill test and increased VO_2max [72]. Kelly et al. reported that NAC ingestion 45 min before submaximal discontinuous cycling exercise reduced exercise-induced respiratory muscle fatigue [73]. Medved et al. administered intravenous NAC in endurance-trained individuals before and during cycling and found an increase in submaximal performance and a 26% increase in the time to exhaustion. They also reported increased muscle cysteine levels and GSH availability in the individuals [74]. However, despite the positive effects of NAC, Rhodes and Braakhuis concluded in their meta-analysis that the side effects of NAC supplementation increased with increasing doses and that it could no longer be recommended [75]. In light of recent studies suggesting the effectiveness of oral glutathione supplementation, further research is warranted to elucidate the potential benefits of oral glutathione intake over NAC supplementation in enhancing intracellular glutathione availability.

The interaction between vitamin Cand glutathione has been well established, suggesting that they play a central role in cellular antioxidant defense systems. They are water-soluble and function in the cytoplasm and mitochondria [47,76-79]. The series of oxidation-reduction reactions that allow vitamin C and glutathione to interact each other makes them unique. The cyclic reactions are shown in Figure 3. The regeneration of the reduced compounds through this interconnected series of reactions amplifies the antioxidant capabilities of each compound. Without this redox coupling, the removal of oxidants would be constrained by the concentration of each compound. Mårtensson and Meister induced glutathione deficiency in adult mice by inhibitor administration. They observed a rapid and substantial increase in ascorbate levels in the liver of mice. Subsequently, the ascorbate levels decreased, whereas dehydroascorbate levels significantly increased. This suggests that glutathione deficiency hinders the reduction of dehydroascorbate, sustaining its elevated levels. Similar trends were observed in the kidneys and lungs, where ascorbate levels decreased, and dehydroascorbate levels increased. Therefore, during oxidative stress, glutathione depletion enhances ascorbate synthesis to protect against oxidative damage [80].

Beyond their biochemical interconnection, the profound association between vitamin C and glutathione is evident through research demonstrating changes in their levels in the blood or tissue upon vitamin C and/or glutathione supplementation. Vitamin C supplementation for 3 weeks in 48 individuals with ascorbate deficiency resulted in an 18% increase in lymphocyte glutathione levels compared with the placebo group [81]. Johnston et al. reported that vitamin C supplementation led to increased plasma glutathione concentration, regardless of the dosage (500 or 2000 mg/day). They concluded that vitamin C supplementation increased plasma GSH levels, thereby enhancing the protective antioxidant effect in the blood [82]. Ascorbate administration to newborn rats with glutathione deficiency has been shown to reduce toxicity and mortality rates and increase tissue and mitochondrial glutathione levels [83].

Despite the association between glutathione and vitamin C, studies investigating their combined intake are scarce. Sastre et al. administered glutathione, vitamin C, or NAC to rats orally for 7 days. In the control group, a significant increase in GSSG was observed during exhaustive exercise; however, the increase in GSSG in the supplementation groups was attenuated. As part of the same study, national-level elite athletes were administered 1 g of glutathione and 2 g of vitamin C orally for 7 days, and changes in serum GSH, GSSG, and the GSSG/GSH ratio were assessed before and after exhaustive exercise. Positive improvements in the GSSG/GSH ratio were observed immediately after exhaustive exercise following the intervention, compared to pre-intervention levels. This is valuable as it is the first report of the effects of combined vitamin C and glutathione supplementation on serum glutathione levels after exercise. However, owing to the small sample size of only 5 participants, the results were not statistically analyzed; moreover, there was no control group. Additionally, this study did not examine parameters other than serum glutathione, making it challenging to precisely ascertain the effects of combined vitamin C and glutathione supplementation [84]. We recently studied the effects of combined glutathione and vitamin C supplementation using an improved study design. We found that compared to vitamin C or glutathione single supplementation, acute vitamin C and glutathione complex supplementation in 12 middle-aged triathletes resulted in decreased carbon dioxide output, decreased heart rate, and positive improvements in skeletal muscle oxygenation during 90 min of cycling exercise at 70% VO_2max, as well as positive improvements in blood and biological antioxidant potential (BAP) and diacron reactive oxygen metabolite(dROM) [85]. Except for the two papers mentioned above, no specific studies on the exercise performance effects of combined vitamin C and glutathione supplementation were found in the database search. However, one study investigated the effect of combined supplementation of vitamin C with NAC and zinc on exercise performance. Alduraywish conducted a study involving 18 male novice volleyball players who were administered a combination of 750 mg NAC (a precursor of glutathione), 5 mg zinc, and 100 mg vitamin C for 7 days. The effects of this supplementation on time-to-exhaustion (TTE), cardiorespiratory fitness (CRF) index, and metabolic indicators were investigated. The results indicated significant improvements in TTE and CRF following one-week supplementation with the combination of substances [83]. Although limited, previous studies have suggested that the combined supplementation of vitamin C and glutathione may exhibit additive effects compared to individual supplementation. Additional research and validation studies are necessary to determine precise dosage recommendations and evaluate the extended impacts across a wider spectrum of athletes and athletic scenarios.