Profound ketosis and cortisol elevation without oxidative stress: safety of an 8-day water- only fast combined with physical exercise in healthy adults

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Methods Six healthy volunteers underwent an 8-day water-only fast. A graded cycle ergometer test to volitional exhaustion was performed at baseline (fed state) and on day 8 of fasting. Serum metabolic (β-hydroxybutyrate [β-HB], cortisol) and redox markers (malondialdehyde [MDA], total oxidant/antioxidant status [TOS/TAS], superoxide dismutase [SOD], thiol groups [SH], vitamins A and E) were measured at rest, 3 minutes, and 1 hour post-exercise. Results Fasting induced profound metabolic reprogramming, characterized by a 15-fold increase in resting β-HB (0.32 to 4.88 mmol/L, P < 0.001) and elevated cortisol (302.9 to 426.2 nmol/L, P < 0.001). Despite this severe metabolic stress, exhaustive exercise in the fasted state did not increase lipid peroxidation; MDA levels remained remarkably stable (effect size 0.04). Protective non-enzymatic defenses actively adapted, evidenced by significant post-exercise increases in SH groups (P = 0.04) and TAS (P = 0.02). Conclusions An 8-day water-only fast induces deep physiological ketosis and hypercortisolemia while fully preserving redox homeostasis. The human organism effectively buffers the oxidative burst of exhaustive exercise during prolonged caloric deprivation, supporting the clinical safety of integrating exercise into fasting protocols. Prolonged fasting Oxidative stress Ketosis Malondialdehyde Mitohormesis Exercise physiology INTRODUCTION Intermittent Fasting (IF) and prolonged water-only fasting have evolved from historical practices into potent clinical interventions for metabolic syndrome, obesity, and type 2 diabetes [ 1 ]. The physiological premise of these protocols is the "metabolic switch"—the transition from glucose utilization to fatty acid oxidation and ketogenesis. This switch triggers adaptive responses, including autophagy upregulation, improved insulin sensitivity, and reduced systemic inflammation [ 2 ]. Simultaneously, regular physical activity is a cornerstone of preventive medicine. This creates a clinical dilemma: Is it safe to combine these two stressors? Evolutionarily, the "thrifty genotype" hypothesis suggests humans must possess mechanisms to preserve function during energy deficits [ 3 ]. However, in modern evidence-based medicine, safety must be proven biochemically. Theoretical concerns regarding exercising during starvation are grounded in bioenergetics. High-intensity exercise induces oxidative stress by increasing electron leakage from the mitochondrial electron transport chain, generating reactive oxygen species (ROS) [ 4 ]. Under fed conditions, this acts as a hormetic signal ("mitohormesis") [ 5 ]. However, an 8-day water fast alters the metabolic background, characterized by glycogen depletion and elevated cortisol. Critics argue that superimposing the oxidative burst of exhaustive exercise upon this catabolic environment could overwhelm antioxidant defenses, leading to uncompensated oxidative distress rather than adaptation [ 6 ]. Data on the redox safety of this specific "dual-stressor" model (prolonged starvation plus exhaustive effort) are scarce. Most research focuses on shorter fasting windows or low-intensity activity. In this pilot study, we evaluated a radical intervention: an 8-day water-only fast followed by a graded exercise test to volitional exhaustion. We hypothesized that the healthy human organism retains sufficient plasticity—via ketone-mediated signaling—to maintain homeostasis even under such extreme demands. PATIENTS AND METHODS Study Design and Participants At the beginning of experiment, age, body weight (BW), height and body mass index (BMI) of volunteers were recorded. This prospective, single-arm study involved six healthy volunteers (4 men, 2 women; mean age 55.3(11.2) years. The protocol was approved by the Research Ethics Committee of the Jan Długosz University in Częstochowa (No. KE-U/9/2024) and adhered to the Declaration of Helsinki. All participants provided written informed consent. Exclusion criteria included: history of cardiovascular events, diabetes, renal/hepatic insufficiency, eating disorders, BMI < 18.5 kg/m², or recent antioxidant supplementation. Fasting Protocol Participants underwent an 8-day complete water-only fast (0 kcal/day). Mineral water was consumed ad libitum (min. 2.0 L/day) to support renal clearance of ketone bodies. Medical supervision included daily monitoring of vitals and glucose to prevent adverse events like hypoglycemia. Exercise Challenge A graded exercise test (GXT) was performed on a cycle ergometer at two time points: Baseline: Fed state. Post-Fast: On the 8th day of fasting. Workload started at 30 W, increasing by 30 W every 3 minutes until volitional exhaustion. This endpoint ensured maximal physiological stress in both trials. Biochemical Analysis Peripheral blood samples were collected using a vacuum system. Then, the blood was centrifuged (1500 x g, 15 min). The serum obtained in this way was frozen at -80°C. Venous blood was collected at rest, 3 minutes post-exercise (peak stress), and 1 hour post-exercise (recovery). Metabolic: Serum β-hydroxybutyrate (β-HB) and Cortisol. Serum C and β-HB concentrations were measured using tests from Cortisol-CLIA (SNIBE Co., Ltd., Shenzhen, China) and the RANBUD kit (Randox, Laboratories Ltd., Crumling, UK). Lipid Peroxidation: Malondialdehyde (MDA) via TBARS method [ 7 ]. Enzymatic Defense: Superoxide Dismutase (SOD) activity [ 8 ]. Non-enzymatic Defense: Thiol groups (SH) [ 9 ], Vitamin A and E [ 10 ]. Global Status: Total Oxidant Status (TOS) [ 11 ] and Total Antioxidant Status (TAS) [ 12 ]. Statistical Analysis Data are presented as mean (SD). Differences between baseline and post-fast resting values, including BW, BMI, and metabolic markers (Table 1 , left panel) were analyzed using the Wilcoxon signed-rank test. The effect of exercise and recovery in the fasted state (Table 1 , right panel) was assessed using Friedman's rank analysis of variance with repeated measures and Dunn-Bonferroni post hoc test, using the Statistica 13.3 (TIBCO Software Inc.). Exact P values are reported to two decimal places (or three if P < 0.01), except for values less than 0.001 which are designated as P < 0.001. To assess clinical significance independent of sample size (N = 6), Effect Sizes (ES) were calculated using Cohen’s d (thresholds: 0.2 small, 0.5 medium, 0.8 large). Raw serum concentrations were used to provide a conservative safety assessment. Additionally, a post-hoc power analysis (1-β) was performed for the primary metabolic and antioxidant outcomes to evaluate the robustness of the findings despite the small sample size. Given the exceptionally large effect sizes observed, the sample size of six volunteers provided sufficient statistical power (> 80% to > 99%) to detect primary metabolic adaptations (β-HB, cortisol) and TAS at an α level of 0.05. Table 1 Metabolic efficacy and redox safety profile (N = 6, Mean ± SD). PARAMETER BASELINE (REST) POST-FAST (REST) P-VALUE FAST-ES (Cohen’s d, Power) BASELINE (3' POST-EX) POST-FAST (3' POST-EX) BASELINE (1H REC) POST-FAST (1H REC) P-VALUE EX-ES (Cohen’s d, Power) METABOLIC WEIGHT, kg 77.3 (16.3) 71.0 (15.0) 99%) a - - - - - - BMI, kg/m² 24.9 (4.5) 22.9 (4.1) 99%) a - - - - - - β-HB, mmol/L 0.32 (0.14) 4.88 (0.40) 99%) - - - - - - CORTISOL, nmol/L 302.9 (100) 426.2 (71.9) < 0.001 1.41 (80%) - - - - - - REDOX MDA, µmol/L 2.94 (0.45) 2.74 (0.70) 0.46 0.34 (low)ᵇ 3.31 (0.74) 2.77 (0.64) 3.30 (0.53) 2.13 (0.41) 0.87 0.04 (low)ᵇ TOS, µmol/L 8.96 (1.92) 8.62 (3.75) 0.81 0.11 (low)ᵇ 10.34 (2.32) 7.93 (3.26) 10.09 (4.90) 8.78 (4.18) 0.49 0.20 (low)ᵇ SOD, NU/mL 15.86 (2.50) 14.95 (1.80) 0.36 0.41 (low)ᵇ 16.64 (1.21) 15.37 (1.74) 16.33 (1.50) 16.58 (3.90) 0.40 0.23 (low)ᵇ SH, µmol/L 305.4 (48.0) 334.9 (72.0) 0.31 0.48 (15%) 365.2 (45.1) 386.5 (74.9) 297.9 (79.0) 300.7 (46.0) 0.04 0.70 (28%) TAS, µmol/L 1.28 (0.16) 1.63 (0.24) 0.009 1.70 (94%) 1.28 (0.21) 1.73 (0.21) 1.25 (0.22) 1.52 (0.40) 0.02 0.45 (14%) VIT A, µmol/L 0.46 (0.14) 0.25 (0.14) 0.008 1.50 (86%) 0.43 (0.13) 0.21 (0.12) 0.41 (0.14) 0.20 (0.12) 0.03 0.30 (low)ᵇ VIT E, µmol/L 11.32 (4.90) 10.14 (2.30) 0.53 0.30 (low)ᵇ 10.51 (3.98) 10.00 (2.92) 10.35 (5.20) 9.97 (2.33) 0.85 0.05 (low)ᵇ Effect sizes (Cohen's d) and corresponding post-hoc statistical power (1-β) are shown for the fasting intervention (Fast-ES) and the acute exercise challenge (Ex-ES). ᵃ For Weight and BMI, the extreme significance (P < 0.001) reflects a 100% consistent directional change across all subjects, representing maximum practical power despite large group variance. ᵇ Low post-hoc power for stable redox markers (d < 0.45) confirms the maintenance of redox homeostasis without detectable oxidative distress, expected in pilot safety parameters. Abbreviations: β-HB, β-hydroxybutyrate; BMI, body mass index; Ex-ES, effect size for exercise (Post-Fast (Rest) vs. Post-Fast (3' Post-Ex)); Fast-ES, effect size for fasting (Baseline (Rest) vs. Post-Fast (Rest)); MDA, malondialdehyde; Post-Ex, post-exercise; Rec, recovery; SH, thiol groups; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status; Vit, vitamin. Data are presented as mean ± SD. RESULTS Metabolic Efficacy The fast induced profound metabolic reprogramming. Mean body weight dropped from 77.3(16.3) kg to 71.0(15.0) kg (P < 0.001). Resting serum β-HB levels surged fifteen-fold from 0.32 to 4.88 mmol/L (P < 0.001), with a massive effect size (Fast-ES Cohen’s d = 15.20). Resting cortisol increased from 302.9 to 426.2 nmol/L (P < 0.001; Cohen’s d = 1.41), confirming HPA axis activation [ 13 ]. Redox Safety Profile Despite the metabolic stress, oxidative damage markers remained stable (Table 1 ). Lipid Peroxidation (MDA): No significant increase occurred after fasting (P = 0.46) or exercise (P = 0.87). The effect size for exercise-induced MDA changes in the fasted state was negligible (Ex-ES Cohen’s d = 0.04). Global Balance: TOS remained stable. TAS increased significantly post-exercise in the fasted state (P = 0.02), suggesting antioxidant mobilization. Defenses: SOD remained stable. Thiol groups showed a moderate increase post-exercise (Cohen’s d = 0.70, P = 0.04). Vitamins: Vitamin E was unchanged. Vitamin A significantly decreased 1 hour post-exercise in the fasted group (P = 0.03; Cohen’s d = 0.30). DISCUSSION The Physiological Paradox of the "Fasting Athlete" This study addresses a fundamental question in metabolic physiology: can the human body withstand the synergistic burden of two potent stressors—prolonged caloric deprivation and exhaustive physical exertion—without succumbing to oxidative distress? Our findings present a physiological paradox. An 8-day water-only fast induced a catabolic state characterized by profound hypoglycemia-sparing ketosis and significant hypercortisolemia. Under standard biochemical dogma, superimposing high-intensity exercise on such a glycogen-depleted, stress-hormone-laden background should precipitate a crisis of redox homeostasis, leading to extensive lipid peroxidation and cellular damage [ 4 , 6 ]. However, our data demonstrate the opposite: the maintenance of redox stability and a specific, targeted upregulation of antioxidant defenses. This suggests that the human organism retains an evolutionary "metabolic flexibility" that allows it to uncouple energetic stress from oxidative damage, a trait likely selected to support foraging activity during periods of food scarcity [ 3 ]. Metabolic Reprogramming: Beyond Simple Starvation The magnitude of the metabolic shift observed in our participants confirms strict adherence to the protocol and sets the biochemical stage for interpreting the redox data. The fifteen-fold increase in β-HB to nearly 5 mmol/L represents a state of "deep physiological ketosis," distinct from the pathological ketoacidosis seen in uncontrolled diabetes, yet significantly higher than levels achieved through ketogenic diets or short-term intermittent fasting [ 14 ]. This massive influx of ketone bodies is not merely an alternative fuel source; it represents a fundamental change in cellular signaling. The concomitant rise in cortisol (P < 0.001, Cohen’s d = 1.41) aligns with the "glucocorticoid paradox" of fasting. Unlike the chronic, maladaptive hypercortisolemia associated with psychological stress or metabolic syndrome, fasting-induced cortisol elevation serves a functional, lipolytic purpose [ 13 ]. It mobilizes free fatty acids and amino acids for gluconeogenesis to protect brain function. Crucially, our data show that this catabolic hormone surge did not correlate with increased oxidative damage (MDA), suggesting that the "stress" of fasting is distinct from the "distress" of oxidative injury. Lipid Peroxidation: The "Silent" Marker The most striking finding of this study is the behavior of Malondialdehyde (MDA), a marker of lipid peroxidation. During high-intensity exercise, particularly in untrained or moderately trained individuals, the rapid increase in oxygen flux through the mitochondrial electron transport chain typically increases electron leakage, forming superoxide radicals [ 5 ]. These radicals attack polyunsaturated fatty acids in cell membranes, causing an MDA spike. In our study, despite the exhaustive nature of the test (cycling to volitional failure), MDA levels remained remarkably stable (Cohen’s d = 0.04). We hypothesize two mechanisms for this protection. First, the shift from glucose to ketone body oxidation increases the hydraulic efficiency of the mitochondrial engine. The combustion of β-HB increases the span of the redox potential between Complex I and ubiquinone, which thermodynamically reduces the probability of superoxide generation compared to glycolytic flux [ 14 ]. Second, the composition of the mitochondrial membrane itself may be altered during fasting, making it less susceptible to peroxidative attack, although this would require muscle biopsy verification. The absence of an MDA rise provides robust evidence that the "oxidative burst" of exercise was fully buffered by the endogenous antioxidant systems. Ketones as Molecular Shields and Epigenetic Modulators To explain the resilience of our subjects, we must look beyond simple stoichiometry to cell signaling. β-HB is now recognized as a potent signaling metabolite that acts as an endogenous histone deacetylase (HDAC) inhibitor [ 15 ]. By inhibiting HDACs (specifically classes I and IIa), β-HB promotes histone hyperacetylation at the promoter regions of genes encoding oxidative stress resistance, such as FoxO3a (Forkhead box O3). Activation of FoxO3a triggers the transcription of manganese superoxide dismutase (MnSOD) and catalase [ 16 ]. Although we did not observe a significant increase in serum SOD activity (P = 0.36), this does not contradict the hypothesis. Serum enzymes reflect a "spillover" from tissues or extracellular buffering, whereas the primary upregulation likely occurred intracellularly (mitohormesis) [ 17 ]. Furthermore, deep ketosis activates the hydroxycarboxylic acid receptor 2 (HCAR2) and the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway. Nrf2 is the "master regulator" of the antioxidant response. We propose that the 8-day fast acted as a hormetic stressor, "priming" the Nrf2 pathway so that when the acute stress of exercise occurred, the cellular defense machinery was already in a state of high alert, instantly neutralizing ROS without the need for a delayed systemic enzyme spike. Thiols: The First Line of Defense While enzymatic defenses (SOD) remained stable, the non-enzymatic sector showed dynamic adaptability. The significant increase in thiol groups (SH) post-exercise (P = 0.04, Cohen’s d = 0.70) is a pivotal finding. Thiol groups, primarily found on serum albumin (Cys34 residue) and glutathione, act as "sacrificial antioxidants." They neutralize reactive species by becoming oxidized themselves, thereby sparing vital lipids and DNA [ 9 ]. In the fed state, exhaustive exercise can sometimes deplete SH groups as they are consumed by the ROS flood. In our fasted subjects, the increase in SH levels post-exercise suggests an overcompensatory mobilization from tissue reserves or a shift in the redox potential of the plasma albumin pool. This indicates an active, rather than passive, response to stress. The body, sensing the "double threat" of starvation and exertion, appears to prioritize the integrity of the intravascular antioxidant barrier. The Retinoid Mystery: Mobilization vs. Depletion The behavior of exogenous antioxidants provides further insight into substrate utilization. Vitamin E (tocopherol), which is lipophilic and protects cell membranes, remained stable (P = 0.53). This mirrors the stability of MDA, confirming that membrane lipids were well-protected. In contrast, Vitamin A (retinol) significantly decreased one hour post-exercise (P = 0.03, Cohen’s d = 0.30). This decline should not be interpreted as a deficiency. Retinol is stored primarily in the liver (stellate cells) and is transported in the blood bound to Retinol Binding Protein (RBP). We hypothesize that the observed drop reflects a rapid, demand-driven uptake of retinol by peripheral tissues (skeletal muscle) to support the intense metabolic demand [ 18 ]. Vitamin A derivatives (retinoic acid) are essential for gene expression related to cell repair and differentiation. In elite athletes, exercise has been shown to accelerate retinol turnover [ 19 ]. In the context of our study, the fasting state may have amplified this demand, or conversely, the liver's ability to re-secrete RBP may have been temporarily prioritized toward gluconeogenic tasks. This "mobilization theory" aligns with the adaptive nature of the other observed parameters. Clinical Implications and Safety The clinical implications of these findings are substantial. Intermittent and prolonged fasting are increasingly prescribed for obesity, type 2 diabetes, and as an adjuvant in chemotherapy (to reduce somatic toxicity). A common concern among clinicians is whether patients on such protocols should avoid physical activity to "conserve energy" or prevent "stress overload." Our data challenge the view that rest is mandatory during fasting. On the contrary, the combination of fasting and exercise may offer synergistic benefits. The "Effect Size" analysis reveals that while the metabolic intervention was powerful (Cohen’s d > 15), the oxidative risk was negligible (Cohen’s d < 0.1). This favorable safety profile suggests that supervised, graded exercise can be safely integrated into fasting protocols for healthy adults. This is consistent with previous reports showing that even in clinical populations, caloric restriction protocols do not exacerbate oxidative stress but may rather improve systemic antioxidant capacity [ 20 ]. Furthermore, our previous research on other metabolic stress models, such as experimental diabetes, has shown that antioxidant support can effectively stabilize redox markers like MDA and improve total antioxidant status [ 21 ]. Finally, our recent findings from this 8-day fasting model confirm that these metabolic and redox adaptations are accompanied by a controlled and safe modulation of the cytokine profile [ 22 ]. However, the dramatic rise in cortisol and fall in Vitamin A warrant monitoring, particularly in populations with adrenal insufficiency or marginal nutritional status. Limitations and Future Directions Despite the robust internal validity provided by the repeated-measures design, this pilot study has limitations regarding sample size (N = 6). However, our post-hoc power analysis demonstrates that the study was adequately powered (> 80%) for its primary scientific focus—the metabolic switch and antioxidant mobilization. It remains a pilot assessment for secondary redox markers where effect sizes were inherently negligible, supporting the safety profile of the intervention. The lack of a "fed control group" performing the same exercise protocol is a design choice; our baseline served as the control, but a parallel arm would strengthen the conclusions regarding the specific "fasting effect" versus the "exercise effect." Additionally, we relied on circulating serum markers. While serum redox status is a clinically relevant proxy, it does not perfectly reflect the intramuscular environment. Future research should include muscle biopsies to assess mitochondrial respiration states and specific protein expression (e.g., PGC-1α, UCP3) to definitively prove the mitohormesis hypothesis. CONCLUSION In conclusion, an 8-day water-only fast creates a unique metabolic environment where extreme ketosis and hypercortisolemia coexist with preserved redox homeostasis. The human body appears to effectively uncouple the catabolic stress of starvation from oxidative cellular damage, even when challenged with exhaustive physical work. This resilience is likely mediated by a multi-layered defense system: the thermodynamic efficiency of ketone bodies, the signaling properties of β-HB (HDAC inhibition), and the active mobilization of thiol-based antioxidant barriers. These findings support the safety of combining caloric restriction with physical activity and provide a biochemical rationale for the evolutionary success of the "hungry, active human." Declarations Funding: This research was financed from the statutory funds of the Jan Dlugosz University in Czestochowa (Poland). Competing interests: The authors declare no competing interests. Author contributions: Conceptualization, P.B. and W.P.; methodology, P.B. and P.D.; software, P.D.; validation, P.B. and P.D.; formal analysis, J.Z-F. and P.D.; investigation, P.B. and W.P.; resources, P.B.; data curation, P.B. and P.D.; writing—original draft preparation, P.B. and W.P.; writing—review and editing, W.P. and J.Z-F.; visualization, P.B.; supervision, W.P. and J.Z-F.; project administration, P.B. All authors edited and approved the final version of the manuscript. Human Ethics: The study protocol was approved by the Research Ethics Committee of the Jan Dlugosz University in Częstochowa (No. KE-U/9/2024) and adhered to the principles of the Declaration of Helsinki. Consent to Participate: Written informed consent was obtained from all individual participants included in the study. Clinical trial number: not applicable. Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements: The authors would like to thank all study participants who contributed their time to this project. References de Cabo R, Mattson MP. Effects of Intermittent Fasting on Health, Aging, and Disease. N Engl J Med. 2019; 381: 2541–2551. Wilhelmi de Toledo F, Grundler F, Bergouignan A, et al. Safety, health improvement and well-being during a 4 to 21-day fasting period in an observational study including 1422 subjects. PLoS One. 2019; 14: e0209353. Cahill GF Jr. Starvation in man. N Engl J Med. 1970; 282: 668–675. Powers SK, Jackson MJ. Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production. Physiol Rev. 2008; 88: 1243–1276. Fisher-Wellman K, Bloomer RJ. Acute exercise and oxidative stress: a 30 year history. Dyn Med. 2009; 8: 1. Radak Z, Chung HY, Goto S. 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J Anim Physiol Anim Nutr (Berl). 2004; 88: 234–238. Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med. 2007; 43: 4–15. Johnson JB, Summer W, Cutler RG, et al. Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Free Radic Biol Med. 2007; 42: 665–674. Bramora P, Zych M, Borymska W, Kaczmarczyk-Sedlak I. Effect of silymarin on the parameters of oxidative stress in hearts in the course of diabetes mellitus in Wistar rats. Acta Pol Pharm. 2022; 79: 901–911. Bramora P, Dolibog P, Zalejska-Fiolka J, et al. The effect of fasting and physical exercise on serum levels of selected cytokines in middle-aged individuals – pilot study. Arch Budo. 2025; 21: 130–139. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9556152","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632006022,"identity":"8b686818-3f4c-49ae-9f47-c32594e12a8b","order_by":0,"name":"Piotr Jan Bramora","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYFACHijN3sBgAGQzNgDxAeK08ByAa2EgUotEAphNWIs5A++xDz9zbPL5Z74xKHjDYCO74QDvAbxaLBv4kmf2bkuznHE7x8BwDkOa8YYDfAl4tRgc4DFm4N122IABqMWYh+Fw4oYDPAYEtTD+BWqRv3kGpOU/cVqYQbYY3OABaTlAWItlM18ys+y2NAPDM2kFhnMMko1nHibgF3P23sOMb7fZGMgdP7zN4E2FnWzf8d6DD/A6jBnBZjNgMABSzDw4VUO0ILGZoYYT0DIKRsEoGAUjDgAAfdVKJhtmT50AAAAASUVORK5CYII=","orcid":"","institution":"W. Bieganski Collegium Medicum Jan Dlugosz University","correspondingAuthor":true,"prefix":"","firstName":"Piotr","middleName":"Jan","lastName":"Bramora","suffix":""},{"id":632006024,"identity":"4d69d129-a41d-4e7b-8594-f4b6be8c3ec2","order_by":1,"name":"Paweł Dolibog","email":"","orcid":"","institution":"Medical University of Silesia","correspondingAuthor":false,"prefix":"","firstName":"Paweł","middleName":"","lastName":"Dolibog","suffix":""},{"id":632006025,"identity":"353e1df4-f93e-41c9-ac73-42f762a066d9","order_by":2,"name":"Jolanta Zalejska-Fiolka","email":"","orcid":"","institution":"Medical University of Silesia","correspondingAuthor":false,"prefix":"","firstName":"Jolanta","middleName":"","lastName":"Zalejska-Fiolka","suffix":""},{"id":632006026,"identity":"e7d51e6f-38ea-47b0-a43d-d3166a510e57","order_by":3,"name":"Wiesław Pilis","email":"","orcid":"","institution":"W. Bieganski Collegium Medicum Jan Dlugosz University","correspondingAuthor":false,"prefix":"","firstName":"Wiesław","middleName":"","lastName":"Pilis","suffix":""}],"badges":[],"createdAt":"2026-04-28 15:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9556152/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9556152/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109281325,"identity":"2ca0968b-840b-484e-b4ac-92d4cff4a163","added_by":"auto","created_at":"2026-05-14 18:00:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":205499,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9556152/v1/c4bd9afd-7491-40eb-8d9e-b479bd9cefb8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Profound ketosis and cortisol elevation without oxidative stress: safety of an 8-day water- only fast combined with physical exercise in healthy adults","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIntermittent Fasting (IF) and prolonged water-only fasting have evolved from historical practices into potent clinical interventions for metabolic syndrome, obesity, and type 2 diabetes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The physiological premise of these protocols is the \"metabolic switch\"\u0026mdash;the transition from glucose utilization to fatty acid oxidation and ketogenesis. This switch triggers adaptive responses, including autophagy upregulation, improved insulin sensitivity, and reduced systemic inflammation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimultaneously, regular physical activity is a cornerstone of preventive medicine. This creates a clinical dilemma: Is it safe to combine these two stressors? Evolutionarily, the \"thrifty genotype\" hypothesis suggests humans must possess mechanisms to preserve function during energy deficits [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, in modern evidence-based medicine, safety must be proven biochemically.\u003c/p\u003e \u003cp\u003eTheoretical concerns regarding exercising during starvation are grounded in bioenergetics. High-intensity exercise induces oxidative stress by increasing electron leakage from the mitochondrial electron transport chain, generating reactive oxygen species (ROS) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Under fed conditions, this acts as a hormetic signal (\"mitohormesis\") [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, an 8-day water fast alters the metabolic background, characterized by glycogen depletion and elevated cortisol. Critics argue that superimposing the oxidative burst of exhaustive exercise upon this catabolic environment could overwhelm antioxidant defenses, leading to uncompensated oxidative distress rather than adaptation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eData on the redox safety of this specific \"dual-stressor\" model (prolonged starvation plus exhaustive effort) are scarce. Most research focuses on shorter fasting windows or low-intensity activity. In this pilot study, we evaluated a radical intervention: an 8-day water-only fast followed by a graded exercise test to volitional exhaustion. We hypothesized that the healthy human organism retains sufficient plasticity\u0026mdash;via ketone-mediated signaling\u0026mdash;to maintain homeostasis even under such extreme demands.\u003c/p\u003e"},{"header":"PATIENTS AND METHODS","content":"\u003cp\u003eStudy Design and Participants\u003c/p\u003e \u003cp\u003eAt the beginning of experiment, age, body weight (BW), height and body mass index (BMI) of volunteers were recorded. This prospective, single-arm study involved six healthy volunteers (4 men, 2 women; mean age 55.3(11.2) years. The protocol was approved by the Research Ethics Committee of the Jan Długosz University in Częstochowa (No. KE-U/9/2024) and adhered to the Declaration of Helsinki. All participants provided written informed consent. Exclusion criteria included: history of cardiovascular events, diabetes, renal/hepatic insufficiency, eating disorders, BMI\u0026thinsp;\u0026lt;\u0026thinsp;18.5 kg/m\u0026sup2;, or recent antioxidant supplementation.\u003c/p\u003e \u003cp\u003eFasting Protocol\u003c/p\u003e \u003cp\u003eParticipants underwent an 8-day complete water-only fast (0 kcal/day). Mineral water was consumed ad libitum (min. 2.0 L/day) to support renal clearance of ketone bodies. Medical supervision included daily monitoring of vitals and glucose to prevent adverse events like hypoglycemia.\u003c/p\u003e \u003cp\u003eExercise Challenge\u003c/p\u003e \u003cp\u003eA graded exercise test (GXT) was performed on a cycle ergometer at two time points:\u003c/p\u003e \u003cp\u003eBaseline: Fed state.\u003c/p\u003e \u003cp\u003ePost-Fast: On the 8th day of fasting. Workload started at 30 W, increasing by 30 W every 3 minutes until volitional exhaustion. This endpoint ensured maximal physiological stress in both trials.\u003c/p\u003e \u003cp\u003eBiochemical Analysis\u003c/p\u003e \u003cp\u003ePeripheral blood samples were collected using a vacuum system. Then, the blood was centrifuged (1500 x g, 15 min). The serum obtained in this way was frozen at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eVenous blood was collected at rest, 3 minutes post-exercise (peak stress), and 1 hour post-exercise (recovery).\u003c/p\u003e \u003cp\u003eMetabolic: Serum β-hydroxybutyrate (β-HB) and Cortisol. Serum C and β-HB concentrations were measured using tests from Cortisol-CLIA (SNIBE Co., Ltd., Shenzhen, China) and the RANBUD kit (Randox, Laboratories Ltd., Crumling, UK).\u003c/p\u003e \u003cp\u003eLipid Peroxidation: Malondialdehyde (MDA) via TBARS method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzymatic Defense: Superoxide Dismutase (SOD) activity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNon-enzymatic Defense: Thiol groups (SH) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Vitamin A and E [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlobal Status: Total Oxidant Status (TOS) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Total Antioxidant Status (TAS) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean (SD). Differences between baseline and post-fast resting values, including BW, BMI, and metabolic markers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel) were analyzed using the Wilcoxon signed-rank test. The effect of exercise and recovery in the fasted state (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, right panel) was assessed using Friedman's rank analysis of variance with repeated measures and Dunn-Bonferroni post hoc test, using the Statistica 13.3 (TIBCO Software Inc.). Exact P values are reported to two decimal places (or three if P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), except for values less than 0.001 which are designated as P\u0026thinsp;\u0026lt;\u0026thinsp;0.001. To assess clinical significance independent of sample size (N\u0026thinsp;=\u0026thinsp;6), Effect Sizes (ES) were calculated using Cohen\u0026rsquo;s d (thresholds: 0.2 small, 0.5 medium, 0.8 large). Raw serum concentrations were used to provide a conservative safety assessment. Additionally, a post-hoc power analysis (1-β) was performed for the primary metabolic and antioxidant outcomes to evaluate the robustness of the findings despite the small sample size. Given the exceptionally large effect sizes observed, the sample size of six volunteers provided sufficient statistical power (\u0026gt;\u0026thinsp;80% to \u0026gt;\u0026thinsp;99%) to detect primary metabolic adaptations (β-HB, cortisol) and TAS at an α level of 0.05.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMetabolic efficacy and redox safety profile (N\u0026thinsp;=\u0026thinsp;6, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePARAMETER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBASELINE (REST)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePOST-FAST (REST)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-VALUE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFAST-ES (Cohen\u0026rsquo;s d, Power)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBASELINE (3' POST-EX)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePOST-FAST (3' POST-EX)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBASELINE (1H REC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePOST-FAST (1H REC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP-VALUE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eEX-ES (Cohen\u0026rsquo;s d, Power)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMETABOLIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWEIGHT, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77.3 (16.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.0 (15.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40 (\u0026gt;\u0026thinsp;99%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI, kg/m\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.9 (4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.9 (4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.48 (\u0026gt;\u0026thinsp;99%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-HB, mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.32 (0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.88 (0.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.20 (\u0026gt;\u0026thinsp;99%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCORTISOL, nmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e302.9 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e426.2 (71.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.41 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eREDOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.94 (0.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.74 (0.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.31 (0.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.77 (0.64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.30 (0.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.13 (0.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.04 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTOS, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.96 (1.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.62 (3.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.11 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.34 (2.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.93 (3.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.09 (4.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.78 (4.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.20 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD, NU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.86 (2.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.95 (1.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.64 (1.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.37 (1.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16.33 (1.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.58 (3.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.23 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSH, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e305.4 (48.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e334.9 (72.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.48 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e365.2 (45.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e386.5 (74.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e297.9 (79.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e300.7 (46.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.70 (28%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTAS, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.28 (0.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.63 (0.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.70 (94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.28 (0.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.73 (0.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.25 (0.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.52 (0.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.45 (14%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVIT A, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.46 (0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.25 (0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.50 (86%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.43 (0.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.21 (0.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.41 (0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.20 (0.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.30 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVIT E, \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.32 (4.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.14 (2.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.51 (3.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.00 (2.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.35 (5.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.97 (2.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.05 (low)ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eEffect sizes (Cohen's d) and corresponding post-hoc statistical power (1-β) are shown for the fasting intervention (Fast-ES) and the acute exercise challenge (Ex-ES).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eᵃ For Weight and BMI, the extreme significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) reflects a 100% consistent directional change across all subjects, representing maximum practical power despite large group variance.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eᵇ Low post-hoc power for stable redox markers (d\u0026thinsp;\u0026lt;\u0026thinsp;0.45) confirms the maintenance of redox homeostasis without detectable oxidative distress, expected in pilot safety parameters.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eAbbreviations: β-HB, β-hydroxybutyrate; BMI, body mass index; Ex-ES, effect size for exercise (Post-Fast (Rest) vs. Post-Fast (3' Post-Ex)); Fast-ES, effect size for fasting (Baseline (Rest) vs. Post-Fast (Rest)); MDA, malondialdehyde; Post-Ex, post-exercise; Rec, recovery; SH, thiol groups; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status; Vit, vitamin. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eMetabolic Efficacy\u003c/p\u003e \u003cp\u003eThe fast induced profound metabolic reprogramming. Mean body weight dropped from 77.3(16.3) kg to 71.0(15.0) kg (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Resting serum β-HB levels surged fifteen-fold from 0.32 to 4.88 mmol/L (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a massive effect size (Fast-ES Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;15.20). Resting cortisol increased from 302.9 to 426.2 nmol/L (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.41), confirming HPA axis activation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRedox Safety Profile\u003c/p\u003e \u003cp\u003eDespite the metabolic stress, oxidative damage markers remained stable (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLipid Peroxidation (MDA): No significant increase occurred after fasting (P\u0026thinsp;=\u0026thinsp;0.46) or exercise (P\u0026thinsp;=\u0026thinsp;0.87). The effect size for exercise-induced MDA changes in the fasted state was negligible (Ex-ES Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e \u003cp\u003eGlobal Balance: TOS remained stable. TAS increased significantly post-exercise in the fasted state (P\u0026thinsp;=\u0026thinsp;0.02), suggesting antioxidant mobilization.\u003c/p\u003e \u003cp\u003eDefenses: SOD remained stable. Thiol groups showed a moderate increase post-exercise (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.70, P\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e \u003cp\u003eVitamins: Vitamin E was unchanged. Vitamin A significantly decreased 1 hour post-exercise in the fasted group (P\u0026thinsp;=\u0026thinsp;0.03; Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.30).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe Physiological Paradox of the \"Fasting Athlete\"\u003c/p\u003e \u003cp\u003eThis study addresses a fundamental question in metabolic physiology: can the human body withstand the synergistic burden of two potent stressors\u0026mdash;prolonged caloric deprivation and exhaustive physical exertion\u0026mdash;without succumbing to oxidative distress? Our findings present a physiological paradox. An 8-day water-only fast induced a catabolic state characterized by profound hypoglycemia-sparing ketosis and significant hypercortisolemia. Under standard biochemical dogma, superimposing high-intensity exercise on such a glycogen-depleted, stress-hormone-laden background should precipitate a crisis of redox homeostasis, leading to extensive lipid peroxidation and cellular damage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, our data demonstrate the opposite: the maintenance of redox stability and a specific, targeted upregulation of antioxidant defenses. This suggests that the human organism retains an evolutionary \"metabolic flexibility\" that allows it to uncouple energetic stress from oxidative damage, a trait likely selected to support foraging activity during periods of food scarcity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetabolic Reprogramming: Beyond Simple Starvation\u003c/p\u003e \u003cp\u003e The magnitude of the metabolic shift observed in our participants confirms strict adherence to the protocol and sets the biochemical stage for interpreting the redox data. The fifteen-fold increase in β-HB to nearly 5 mmol/L represents a state of \"deep physiological ketosis,\" distinct from the pathological ketoacidosis seen in uncontrolled diabetes, yet significantly higher than levels achieved through ketogenic diets or short-term intermittent fasting [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This massive influx of ketone bodies is not merely an alternative fuel source; it represents a fundamental change in cellular signaling. The concomitant rise in cortisol (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.41) aligns with the \"glucocorticoid paradox\" of fasting. Unlike the chronic, maladaptive hypercortisolemia associated with psychological stress or metabolic syndrome, fasting-induced cortisol elevation serves a functional, lipolytic purpose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It mobilizes free fatty acids and amino acids for gluconeogenesis to protect brain function. Crucially, our data show that this catabolic hormone surge did not correlate with increased oxidative damage (MDA), suggesting that the \"stress\" of fasting is distinct from the \"distress\" of oxidative injury.\u003c/p\u003e \u003cp\u003eLipid Peroxidation: The \"Silent\" Marker\u003c/p\u003e \u003cp\u003eThe most striking finding of this study is the behavior of Malondialdehyde (MDA), a marker of lipid peroxidation. During high-intensity exercise, particularly in untrained or moderately trained individuals, the rapid increase in oxygen flux through the mitochondrial electron transport chain typically increases electron leakage, forming superoxide radicals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These radicals attack polyunsaturated fatty acids in cell membranes, causing an MDA spike. In our study, despite the exhaustive nature of the test (cycling to volitional failure), MDA levels remained remarkably stable (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e \u003cp\u003eWe hypothesize two mechanisms for this protection. First, the shift from glucose to ketone body oxidation increases the hydraulic efficiency of the mitochondrial engine. The combustion of β-HB increases the span of the redox potential between Complex I and ubiquinone, which thermodynamically reduces the probability of superoxide generation compared to glycolytic flux [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Second, the composition of the mitochondrial membrane itself may be altered during fasting, making it less susceptible to peroxidative attack, although this would require muscle biopsy verification. The absence of an MDA rise provides robust evidence that the \"oxidative burst\" of exercise was fully buffered by the endogenous antioxidant systems.\u003c/p\u003e \u003cp\u003eKetones as Molecular Shields and Epigenetic Modulators\u003c/p\u003e \u003cp\u003eTo explain the resilience of our subjects, we must look beyond simple stoichiometry to cell signaling. β-HB is now recognized as a potent signaling metabolite that acts as an endogenous histone deacetylase (HDAC) inhibitor [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By inhibiting HDACs (specifically classes I and IIa), β-HB promotes histone hyperacetylation at the promoter regions of genes encoding oxidative stress resistance, such as FoxO3a (Forkhead box O3). Activation of FoxO3a triggers the transcription of manganese superoxide dismutase (MnSOD) and catalase [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough we did not observe a significant increase in serum SOD activity (P\u0026thinsp;=\u0026thinsp;0.36), this does not contradict the hypothesis. Serum enzymes reflect a \"spillover\" from tissues or extracellular buffering, whereas the primary upregulation likely occurred intracellularly (mitohormesis) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, deep ketosis activates the hydroxycarboxylic acid receptor 2 (HCAR2) and the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway. Nrf2 is the \"master regulator\" of the antioxidant response. We propose that the 8-day fast acted as a hormetic stressor, \"priming\" the Nrf2 pathway so that when the acute stress of exercise occurred, the cellular defense machinery was already in a state of high alert, instantly neutralizing ROS without the need for a delayed systemic enzyme spike.\u003c/p\u003e \u003cp\u003eThiols: The First Line of Defense\u003c/p\u003e \u003cp\u003eWhile enzymatic defenses (SOD) remained stable, the non-enzymatic sector showed dynamic adaptability. The significant increase in thiol groups (SH) post-exercise (P\u0026thinsp;=\u0026thinsp;0.04, Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.70) is a pivotal finding. Thiol groups, primarily found on serum albumin (Cys34 residue) and glutathione, act as \"sacrificial antioxidants.\" They neutralize reactive species by becoming oxidized themselves, thereby sparing vital lipids and DNA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the fed state, exhaustive exercise can sometimes deplete SH groups as they are consumed by the ROS flood. In our fasted subjects, the increase in SH levels post-exercise suggests an overcompensatory mobilization from tissue reserves or a shift in the redox potential of the plasma albumin pool. This indicates an active, rather than passive, response to stress. The body, sensing the \"double threat\" of starvation and exertion, appears to prioritize the integrity of the intravascular antioxidant barrier.\u003c/p\u003e \u003cp\u003eThe Retinoid Mystery: Mobilization vs. Depletion\u003c/p\u003e \u003cp\u003eThe behavior of exogenous antioxidants provides further insight into substrate utilization. Vitamin E (tocopherol), which is lipophilic and protects cell membranes, remained stable (P\u0026thinsp;=\u0026thinsp;0.53). This mirrors the stability of MDA, confirming that membrane lipids were well-protected. In contrast, Vitamin A (retinol) significantly decreased one hour post-exercise (P\u0026thinsp;=\u0026thinsp;0.03, Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.30).\u003c/p\u003e \u003cp\u003eThis decline should not be interpreted as a deficiency. Retinol is stored primarily in the liver (stellate cells) and is transported in the blood bound to Retinol Binding Protein (RBP). We hypothesize that the observed drop reflects a rapid, demand-driven uptake of retinol by peripheral tissues (skeletal muscle) to support the intense metabolic demand [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Vitamin A derivatives (retinoic acid) are essential for gene expression related to cell repair and differentiation. In elite athletes, exercise has been shown to accelerate retinol turnover [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the context of our study, the fasting state may have amplified this demand, or conversely, the liver's ability to re-secrete RBP may have been temporarily prioritized toward gluconeogenic tasks. This \"mobilization theory\" aligns with the adaptive nature of the other observed parameters.\u003c/p\u003e \u003cp\u003eClinical Implications and Safety\u003c/p\u003e \u003cp\u003eThe clinical implications of these findings are substantial. Intermittent and prolonged fasting are increasingly prescribed for obesity, type 2 diabetes, and as an adjuvant in chemotherapy (to reduce somatic toxicity). A common concern among clinicians is whether patients on such protocols should avoid physical activity to \"conserve energy\" or prevent \"stress overload.\" Our data challenge the view that rest is mandatory during fasting.\u003c/p\u003e \u003cp\u003eOn the contrary, the combination of fasting and exercise may offer synergistic benefits. The \"Effect Size\" analysis reveals that while the metabolic intervention was powerful (Cohen\u0026rsquo;s d\u0026thinsp;\u0026gt;\u0026thinsp;15), the oxidative risk was negligible (Cohen\u0026rsquo;s d\u0026thinsp;\u0026lt;\u0026thinsp;0.1). This favorable safety profile suggests that supervised, graded exercise can be safely integrated into fasting protocols for healthy adults. This is consistent with previous reports showing that even in clinical populations, caloric restriction protocols do not exacerbate oxidative stress but may rather improve systemic antioxidant capacity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, our previous research on other metabolic stress models, such as experimental diabetes, has shown that antioxidant support can effectively stabilize redox markers like MDA and improve total antioxidant status [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Finally, our recent findings from this 8-day fasting model confirm that these metabolic and redox adaptations are accompanied by a controlled and safe modulation of the cytokine profile [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the dramatic rise in cortisol and fall in Vitamin A warrant monitoring, particularly in populations with adrenal insufficiency or marginal nutritional status.\u003c/p\u003e \u003cp\u003eLimitations and Future Directions\u003c/p\u003e \u003cp\u003eDespite the robust internal validity provided by the repeated-measures design, this pilot study has limitations regarding sample size (N\u0026thinsp;=\u0026thinsp;6). However, our post-hoc power analysis demonstrates that the study was adequately powered (\u0026gt;\u0026thinsp;80%) for its primary scientific focus\u0026mdash;the metabolic switch and antioxidant mobilization. It remains a pilot assessment for secondary redox markers where effect sizes were inherently negligible, supporting the safety profile of the intervention. The lack of a \"fed control group\" performing the same exercise protocol is a design choice; our baseline served as the control, but a parallel arm would strengthen the conclusions regarding the specific \"fasting effect\" versus the \"exercise effect.\" Additionally, we relied on circulating serum markers. While serum redox status is a clinically relevant proxy, it does not perfectly reflect the intramuscular environment. Future research should include muscle biopsies to assess mitochondrial respiration states and specific protein expression (e.g., PGC-1α, UCP3) to definitively prove the mitohormesis hypothesis.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, an 8-day water-only fast creates a unique metabolic environment where extreme ketosis and hypercortisolemia coexist with preserved redox homeostasis. The human body appears to effectively uncouple the catabolic stress of starvation from oxidative cellular damage, even when challenged with exhaustive physical work. This resilience is likely mediated by a multi-layered defense system: the thermodynamic efficiency of ketone bodies, the signaling properties of β-HB (HDAC inhibition), and the active mobilization of thiol-based antioxidant barriers. These findings support the safety of combining caloric restriction with physical activity and provide a biochemical rationale for the evolutionary success of the \"hungry, active human.\"\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding: \u003c/strong\u003eThis research was financed from the statutory funds of the Jan Dlugosz University in Czestochowa (Poland).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: \u003c/strong\u003eConceptualization, P.B. and W.P.; methodology, P.B. and P.D.; software, P.D.; validation, P.B. and P.D.; formal analysis, J.Z-F. and P.D.; investigation, P.B. and W.P.; resources, P.B.; data curation, P.B. and P.D.; writing\u0026mdash;original draft preparation, P.B. and W.P.; writing\u0026mdash;review and editing, W.P. and J.Z-F.; visualization, P.B.; supervision, W.P. and J.Z-F.; project administration, P.B. All authors edited and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Ethics:\u003c/strong\u003e The study protocol was approved by the Research Ethics Committee of the Jan Dlugosz University in Częstochowa (No. KE-U/9/2024) and adhered to the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate: \u003c/strong\u003eWritten informed consent was obtained from all individual participants included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability: \u003c/strong\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u003c/strong\u003eThe authors would like to thank all study participants who contributed their time to this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Cabo R, Mattson MP. Effects of Intermittent Fasting on Health, Aging, and Disease. N Engl J Med. 2019; 381: 2541\u0026ndash;2551.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilhelmi de Toledo F, Grundler F, Bergouignan A, et al. Safety, health improvement and well-being during a 4 to 21-day fasting period in an observational study including 1422 subjects. PLoS One. 2019; 14: e0209353.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCahill GF Jr. Starvation in man. N Engl J Med. 1970; 282: 668\u0026ndash;675.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowers SK, Jackson MJ. Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production. Physiol Rev. 2008; 88: 1243\u0026ndash;1276.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFisher-Wellman K, Bloomer RJ. Acute exercise and oxidative stress: a 30 year history. Dyn Med. 2009; 8: 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadak Z, Chung HY, Goto S. Systemic adaptation to oxidative challenge induced by regular exercise. Free Radic Biol Med. 2008; 44: 153\u0026ndash;159.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979; 95: 351\u0026ndash;358.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal Biochem. 1984; 142: 290\u0026ndash;296.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoster JF, Biemond P, Swaak AJ. Intracellular and extracellular sulphydryl levels in rheumatoid arthritis. Ann Rheum Dis. 1986; 45: 44\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRutkowski M, Grzegorczyk K. Modifications of spectrophotometric methods for antioxidative vitamins determination convenient in analytic practice. Acta Sci Pol Technol Aliment. 2007; 6(3): 17\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005; 38: 1103\u0026ndash;1111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem. 2004; 37: 277\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDjurhuus CB, Gravholt CH, Nielsen S, et al. Effects of cortisol on lipolysis and regional interstitial glycerol levels in humans. Am J Physiol Endocrinol Metab. 2002; 283(1): E172-E177.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewman JC, Verdin E. Beta-Hydroxybutyrate: A Signaling Metabolite. Annu Rev Nutr. 2017; 37: 51\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimazu T, Hirschey MD, Newman J, et al. Suppression of oxidative stress by beta-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science. 2013; 339: 211\u0026ndash;214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRistow M, Zarse K, Oberbach A, et al. Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci U S A. 2009; 106: 8665\u0026ndash;8670.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalabrese V, Cornelius C, Cuzzocrea S, et al. Hormesis, cellular stress response and vitagenes as key players in aging and longevity. J Gerontol A Biol Sci Med Sci. 2012; 67: 157\u0026ndash;167.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaila J, Stohrer M, Forterre S, et al. Effect of exercise on the mobilization of retinol and retinyl esters in plasma of sled dogs. J Anim Physiol Anim Nutr (Berl). 2004; 88: 234\u0026ndash;238.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTraber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med. 2007; 43: 4\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson JB, Summer W, Cutler RG, et al. Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Free Radic Biol Med. 2007; 42: 665\u0026ndash;674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBramora P, Zych M, Borymska W, Kaczmarczyk-Sedlak I. Effect of silymarin on the parameters of oxidative stress in hearts in the course of diabetes mellitus in Wistar rats. Acta Pol Pharm. 2022; 79: 901\u0026ndash;911.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBramora P, Dolibog P, Zalejska-Fiolka J, et al. The effect of fasting and physical exercise on serum levels of selected cytokines in middle-aged individuals \u0026ndash; pilot study. Arch Budo. 2025; 21: 130\u0026ndash;139.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"sport-sciences-for-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ssfh","sideBox":"Learn more about [Sport Sciences for Health](http://link.springer.com/journal/11332)","snPcode":"11332","submissionUrl":"https://submission.nature.com/new-submission/11332/3","title":"Sport Sciences for Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Prolonged fasting, Oxidative stress, Ketosis, Malondialdehyde, Mitohormesis, Exercise physiology","lastPublishedDoi":"10.21203/rs.3.rs-9556152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9556152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo evaluate the redox safety and metabolic effects of superimposing exhaustive physical exercise on a catabolic state induced by an 8-day water-only fast.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSix healthy volunteers underwent an 8-day water-only fast. A graded cycle ergometer test to volitional exhaustion was performed at baseline (fed state) and on day 8 of fasting. Serum metabolic (β-hydroxybutyrate [β-HB], cortisol) and redox markers (malondialdehyde [MDA], total oxidant/antioxidant status [TOS/TAS], superoxide dismutase [SOD], thiol groups [SH], vitamins A and E) were measured at rest, 3 minutes, and 1 hour post-exercise.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFasting induced profound metabolic reprogramming, characterized by a 15-fold increase in resting β-HB (0.32 to 4.88 mmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and elevated cortisol (302.9 to 426.2 nmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Despite this severe metabolic stress, exhaustive exercise in the fasted state did not increase lipid peroxidation; MDA levels remained remarkably stable (effect size 0.04). Protective non-enzymatic defenses actively adapted, evidenced by significant post-exercise increases in SH groups (P\u0026thinsp;=\u0026thinsp;0.04) and TAS (P\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAn 8-day water-only fast induces deep physiological ketosis and hypercortisolemia while fully preserving redox homeostasis. The human organism effectively buffers the oxidative burst of exhaustive exercise during prolonged caloric deprivation, supporting the clinical safety of integrating exercise into fasting protocols.\u003c/p\u003e","manuscriptTitle":"Profound ketosis and cortisol elevation without oxidative stress: safety of an 8-day water- only fast combined with physical exercise in healthy adults","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-14 18:00:10","doi":"10.21203/rs.3.rs-9556152/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T20:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T09:41:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T09:41:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Sport Sciences for Health","date":"2026-04-28T15:24:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"sport-sciences-for-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ssfh","sideBox":"Learn more about [Sport Sciences for Health](http://link.springer.com/journal/11332)","snPcode":"11332","submissionUrl":"https://submission.nature.com/new-submission/11332/3","title":"Sport Sciences for Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a53060ed-d424-4e9b-95a8-fe4a55f71cee","owner":[],"postedDate":"May 14th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-29T20:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T09:41:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T09:41:17+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T18:00:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-14 18:00:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9556152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9556152","identity":"rs-9556152","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}