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This randomized controlled trial investigates the effects of intermittent fasting and water-only fasting on gene expression, tumor markers, quality of life, and chemotherapy tolerance in breast cancer patients receiving chemotherapy. Breast cancer remains the most commonly diagnosed cancer worldwide, and while chemotherapy constitutes standard treatment, it is frequently accompanied by significant adverse effects including cachexia and immunosuppression. Fasting has emerged as a promising complementary approach that may enhance chemotherapy efficacy while protecting healthy cells from treatment-related toxicity. The study will enroll 60 female breast cancer patients from INOR, Abbottabad, who will be randomly assigned to one of four groups: conventional chemotherapy alone, chemotherapy combined with intermittent fasting (23:1 hour fasting to eating ratio) plus routine diet, chemotherapy combined with intermittent fasting plus ketogenic diet, or chemotherapy combined with water-only fasting for two weeks. Blood samples will be collected before and after interventions to assess tumor marker CA 15-3, CD4+ and CD8+ T cell counts, oxidative stress marker malondialdehyde, inflammatory markers including TNF-α and neutrophil-lymphocyte ratio, Glasgow Prognostic Score, and metabolic regulators AMPK and pyruvate dehydrogenase. Quality of life will be evaluated using the validated FACT-B questionnaire, while chemotherapy tolerance will be assessed through symptom severity surveys. The study hypothesizes that combining fasting regimens with standard chemotherapy will enhance anti-tumor effects, improve immune surveillance mechanisms, and ultimately contribute to better prognostic outcomes in breast cancer patients.
This prospective randomized controlled intervention trial is designed to comprehensively evaluate the molecular, immunological, and clinical effects of intermittent and water-only fasting in breast cancer patients undergoing chemotherapy, and will be conducted at the Institute of Nuclear Medicine Oncology and Radiotherapy in Abbottabad in collaboration with the Institute of Basic Medical Sciences, Khyber Medical University, Peshawar. The scientific rationale underlying this investigation stems from the understanding that cancer functions as a metabolic disease characterized by dysfunctional mitochondria and reliance on aerobic glycolysis for energy production, as described by the Warburg hypothesis. By restricting glucose availability through fasting regimens, cancer cells may be selectively starved while normal cells undergo metabolic adaptation, potentially enhancing chemotherapy sensitivity and reducing treatment-related toxicity. Furthermore, fasting has demonstrated immunomodulatory properties through increasing cytotoxic CD8+ T cell precursors and enhancing immune response, while triggering autophagy and inducing differential stress resistance that sensitizes cancer cells to chemotherapy. The study objectives include assessing the effects of intermittent fasting and water-only fasting on quality of life, chemotherapy tolerance, gene expression patterns, and tumor biomarkers, as well as comparing the relative efficacy of these two fasting approaches in improving clinical outcomes. The four study groups will each comprise 15 participants, with group 1 receiving conventional chemotherapy only as the control arm, group 2 undergoing 30 days of intermittent fasting with one routine meal daily, group 3 following 30 days of intermittent fasting with one ketogenic meal daily accompanied by comprehensive dietary guidance and food lists, and group 4 completing a 14-day water-only fast allowing only water, zero-calorie herbal tea, or black coffee without sugar followed by gradual refeeding with juices, broths, and progressive introduction of solid foods. Blood sampling of approximately 10 milliliters will be performed at baseline and following completion of the respective interventions, with serum analyzed for malondialdehyde levels using the thiobarbituric acid method, CD4 and CD8 T cell enumeration through flow cytometry, and quantification of pyruvate dehydrogenase, tumor necrosis factor-alpha, AMPK, albumin, and C-reactive protein using commercially available ELISA kits, while ketone bodies will be measured using a ketone meter. Statistical analysis will be performed using SPSS version 20 with data presented as mean values plus or minus standard error of the mean, analysis of variance applied to identify differences among groups followed by Tukey post-hoc tests, and Pearson correlation coefficients calculated to examine relationships between variables, with statistical significance established at p-values less than 0.05. Ethical approval has been obtained from the Ethics Committee of INOR Abbottabad, and all participants will provide written informed consent after receiving comprehensive information about study procedures, potential benefits, and possible risks including dizziness, weakness, or difficulty tolerating dietary interventions, with assurance of their right to withdraw at any time without providing explanation and without affecting their medical care.
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| Conventional Chemotherapy (Control Group) | Active Comparator | Participants receive conventional chemotherapy treatment according to standard protocols at INOR, Abbottabad, with no dietary restrictions or fasting interventions. Patients consume their routine diet throughout the 4-week study period. Blood samples are collected at baseline and after 4 weeks for biomarker analysis. This group serves as the control arm against which the three fasting intervention groups will be compared. |
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| Intermittent Fasting + Routine Diet | Experimental | Participants receive standard chemotherapy alongside an intermittent fasting regimen consisting of 23 hours of fasting with a 1-hour eating period daily for 30 consecutive days. During the 1-hour eating window, participants consume one routine meal per day consisting of their usual diet without specific dietary modifications. No food or calorie-containing beverages are consumed during the 23-hour fasting period; only water and zero-calorie drinks are permitted. Blood samples are collected at baseline and after completion of the 30-day intervention period. This arm evaluates the effects of intermittent fasting alone without dietary composition changes. |
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| Intermittent Fasting + Ketogenic Diet | Experimental | Participants receive standard chemotherapy combined with an intermittent fasting regimen of 23 hours fasting and 1-hour eating daily for 30 consecutive days. During the 1-hour eating window, participants consume one classic ketogenic meal following provided dietary guidelines. The ketogenic diet comprises high fat content (approximately 75-90% of calories), moderate to low protein, and very low carbohydrates (approximately 5% of calories) to induce and maintain nutritional ketosis. Participants receive comprehensive resources including ketogenic food lists, daily meal plans, and lists of prohibited foods. Only water and zero-calorie beverages are permitted during fasting periods. Blood samples are collected at baseline and post-intervention to assess biomarkers including ketone bodies. This arm evaluates the combined effects of intermittent fasting and ketogenic diet on study outcomes. |
| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| Conventional Chemotherapy | Drug | Participants receive standard chemotherapy treatment according to institutional protocols at INOR. The specific chemotherapy regimen, dosage, frequency, and duration are determined by the treating oncologist based on the patient's histological |
| Measure | Description | Time Frame |
|---|---|---|
| Change in Serum Cancer Antigen 15-3 (CA 15-3) Levels | CA 15-3 is a protein secreted primarily by breast cancer cells and serves as a clinically validated tumor marker for monitoring treatment response and disease progression in breast cancer patients. Blood samples will be collected via venipuncture, and serum CA 15-3 concentrations will be quantified using commercially available ELISA kits according to manufacturer instructions. Measurements will be performed at baseline prior to any intervention and repeated following completion of the assigned fasting protocol. The change in CA 15-3 levels (post-intervention value minus baseline value) will be calculated and compared across the four study groups to evaluate the effect of intermittent fasting, ketogenic diet combined with intermittent fasting, and water-only fasting on this tumor marker relative to conventional chemotherapy alone. | Baseline (prior to intervention initiation) and immediately following completion of the assigned fasting intervention: at 4 weeks for Groups 1, 2, and 3; at 2 weeks for Group 4 (Water-Only Fasting) |
| Change in CD4+ and CD8+ T Cell Counts | CD4+ helper T cells and CD8+ cytotoxic T cells are essential components of adaptive immunity and play critical roles in anti-tumor immune responses. Venous blood samples will be collected at baseline and post-intervention. CD4+ and CD8+ T cell counts will be enumerated using flow cytometry with the FlowCellct Human CD4/CD8 T Cell Kit (MERCK) according to manufacturer protocols. Absolute counts and CD4/CD8 ratios will be determined. Changes in these immune cell populations will be compared across groups to assess whether fasting regimens enhance cellular immunity in breast cancer patients undergoing chemotherapy. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Serum Malondialdehyde (MDA) Levels | Malondialdehyde is a reactive aldehyde and end product of lipid peroxidation, serving as a reliable biomarker of oxidative stress. Elevated MDA levels have been documented in breast cancer patients and correlate with disease progression. Serum MDA concentrations will be determined using the thiobarbituric acid (TBA) method spectrophotometrically. Baseline and post-intervention values will be compared to evaluate whether fasting interventions reduce oxidative stress burden in patients receiving chemotherapy. |
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Inclusion Criteria:
Exclusion Criteria:
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| Name | Affiliation | Role |
|---|---|---|
| Yasmeen Bibi, PhD Scholar | Khyber Medical University Peshawar | Principal Investigator |
| Muhammad Omar Malik, PhD | Khyber Medical University Peshawar, Pakistan | Principal Investigator |
| Sajid Zaman, PhD | Head of Oncology Department, INOR, Abbottabad, Pakistan | Principal Investigator |
| Facility | Status | City | State | ZIP | Country | Contacts |
|---|---|---|---|---|---|---|
| Institute of Nuclear Medicine Oncology & Radiotherapy (INOR) | Abbottābād | Khyber Pakhtunkhwa | 22010 | Pakistan |
| PubMed Identifier | Type | Citation | Retractions |
|---|---|---|---|
| 31817761 | Background | Marquez J, Flores J, Kim AH, Nyamaa B, Nguyen ATT, Park N, Han J. Rescue of TCA Cycle Dysfunction for Cancer Therapy. J Clin Med. 2019 Dec 6;8(12):2161. doi: 10.3390/jcm8122161. | |
| 31937325 | Background | Ghosn B, Benisi-Kohansal S, Ebrahimpour-Koujan S, Azadbakht L, Esmaillzadeh A. Association between healthy lifestyle score and breast cancer. Nutr J. 2020 Jan 14;19(1):4. doi: 10.1186/s12937-020-0520-9. |
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Individual participant data collected during this trial, including de-identified demographic information, clinical characteristics, laboratory biomarker results (CA 15-3, CD4/CD8 counts, MDA, TNF-α, NLR, GPS, AMPK, PDH), quality of life questionnaire responses (FACT-B), and chemotherapy tolerance survey data, will be made available to other researchers upon reasonable request. The data will be shared after de-identification to protect participant privacy. The study protocol, statistical analysis plan, and analytic code will be included to enable reproducibility and secondary analyses.
Individual participant data will become available beginning 6 months after publication of the primary study results and will remain available for a period of 5 years. Data requests will be considered during this timeframe.
Data will be made available to qualified researchers affiliated with academic institutions or research organizations who submit a methodologically sound research proposal. Requests must include a clear description of the proposed analyses, institutional ethics approval or documented exemption, and a signed data sharing agreement that includes: (1) a commitment to use data only for approved research purposes, (2) assurance of no attempts to re-identify participants, (3) agreement to maintain data security, and (4) commitment to acknowledge the original study in any resulting publications. Proposals should be submitted to the Principal Investigator (Dr. Yasmeen Bibi) or the Supervising Investigator (Dr. Muhammad Omar Malik) for review and approval.
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Four parallel groups receiving different dietary interventions alongside standard chemotherapy: control group (chemotherapy only), intermittent fasting with routine diet, intermittent fasting with ketogenic diet, and water-only fasting group.
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Due to the nature of the dietary interventions, participants and study personnel cannot be blinded to group assignment. Participants will be aware of their fasting regimen and dietary restrictions, and investigators will need to provide dietary guidance and monitor compliance. However, laboratory personnel conducting biomarker analyses will be blinded to group allocation to minimize bias in outcome assessment.
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| Water-Only Fasting | Experimental | Participants receive standard chemotherapy combined with a water-only fasting regimen for 14 consecutive days. During this period, participants consume only water, zero-calorie herbal tea, or black coffee without sugar or milk. Limited physical exertion is permitted. After completing the 14-day water-only fast, participants undergo a gradual refeeding protocol beginning with fruit juices and vegetable broths, followed by raw fruits and steamed vegetables, and progressively transitioning back to a normal diet. Blood samples are collected at baseline and after completion of the 14-day fasting period. This arm evaluates the effects of short-term prolonged water-only fasting on tumor markers, immune function, and chemotherapy tolerance in breast cancer patients. |
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| Intermittent Fasting (23:1 Regimen) | Behavioral | Participants undergo an intermittent fasting regimen consisting of 23 consecutive hours of fasting followed by a 1-hour eating period daily for 30 days. During the fasting period, participants consume no food or calorie-containing beverages; only water and zero-calorie drinks (plain water, zero-calorie herbal tea, black coffee without sugar) are permitted. During the 1-hour eating window, participants consume one meal. This regimen is implemented alongside standard chemotherapy. |
|
| Ketogenic Diet | Dietary Supplement | Participants follow a ketogenic diet during their 1-hour eating window as part of the intermittent fasting regimen. The diet consists of high fat content (approximately 75% of calories from fat), moderate to low protein (approximately 20% of calories), and very low carbohydrates (approximately 5% of calories). Participants receive comprehensive dietary resources including ketogenic food lists (meat, fish, eggs, nuts, high-fat dairy, above-ground vegetables, berries, and natural fats such as coconut oil and olive oil), daily meal plans, and lists of prohibited high-carbohydrate foods. This dietary intervention aims to induce and maintain nutritional ketosis throughout the 30-day study period. |
|
| Water-Only Fasting | Behavioral | Participants undergo a water-only fasting regimen for 14 consecutive days. During this period, participants consume only water, zero-calorie herbal tea, or black coffee without sugar or milk. No food or calorie-containing beverages are permitted. Limited physical exertion is advised. Following completion of the 14-day fast, participants undergo a structured gradual refeeding protocol: initial introduction of fruit juices and vegetable broths, followed by raw fruits and steamed vegetables, and progressively transitioning back to a normal diet. This intervention is administered alongside standard chemotherapy. |
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| Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Neutrophil-Lymphocyte Ratio (NLR) | The neutrophil-lymphocyte ratio is a systemic inflammatory marker calculated by dividing absolute neutrophil count by absolute lymphocyte count from complete blood count analysis. Elevated NLR has been associated with poor prognosis in various cancers, including breast cancer. NLR will be calculated from routine complete blood count performed at baseline and post-intervention. Changes in NLR will be analyzed to determine whether fasting regimens modulate systemic inflammation in the context of chemotherapy. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Serum Tumor Necrosis Factor-Alpha (TNF-α) Levels | TNF-α is a pro-inflammatory cytokine involved in systemic inflammation and has been implicated in tumor growth, metastasis, and cancer-related cachexia. Elevated TNF-α levels correlate with advanced disease stage in breast cancer. Serum TNF-α concentrations will be measured using commercially available ELISA kits. Baseline and post-intervention values will be compared across study groups to assess the anti-inflammatory effects of fasting interventions. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Glasgow Prognostic Score (GPS) | The Glasgow Prognostic Score is an inflammation-based prognostic score calculated from serum C-reactive protein (CRP) and albumin levels. The modified GPS used in this study assigns scores as follows: 0 for CRP ≤ 1.0 mg/dl; 1 for CRP > 1.0 mg/dl and albumin ≥ 3.5 mg/dl; 2 for CRP > 1.0 mg/dl and albumin < 3.5 mg/dl. CRP will be measured by immunoassay and albumin by standard laboratory methods. Changes in GPS will be compared to evaluate whether fasting improves the inflammatory and nutritional status of breast cancer patients. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Serum AMP-Activated Protein Kinase (AMPK) Levels | AMPK is a cellular energy sensor activated during glucose shortage and conditions of metabolic stress. Activated AMPK promotes catabolic pathways including autophagy and fatty acid oxidation while inhibiting anabolic processes. Fasting is known to activate AMPK, which may contribute to its anti-tumor effects. Serum AMPK levels will be quantified using ELISA kits. Baseline and post-intervention values will be compared to assess the molecular impact of fasting regimens on cellular energy sensing pathways. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Serum Pyruvate Dehydrogenase (PDH) Levels | Pyruvate dehydrogenase is a mitochondrial enzyme that catalyzes the irreversible conversion of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid cycle. PDH activity serves as a marker of aerobic metabolism and is often dysregulated in cancer cells exhibiting the Warburg effect. Serum PDH levels will be measured using ELISA kits. Changes in PDH will be evaluated to determine whether fasting interventions promote metabolic reprogramming away from aerobic glycolysis toward oxidative phosphorylation. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Quality of Life Assessed by FACT-B Scale | Quality of life will be assessed using the Functional Assessment of Cancer Therapy - Breast Cancer (FACT-B) questionnaire, a validated 37-item instrument consisting of the general FACT-G scale and a breast cancer subscale (BCS). The questionnaire evaluates five domains: physical well-being, social/family well-being, emotional well-being, functional well-being, and breast cancer-specific concerns. Each item is scored from 0 to 4, with higher scores indicating better quality of life. Total scores range from 0 to 148. The questionnaire will be administered at baseline and post-intervention to evaluate whether fasting interventions impact patient-reported quality of life during chemotherapy. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Chemotherapy Tolerance Assessed by Symptom Severity Survey | Chemotherapy tolerance will be evaluated using a symptom severity survey adapted from published fasting and cancer treatment studies. The survey assesses common chemotherapy-related symptoms including fatigue, nausea, vomiting, appetite changes, taste alterations, dry mouth, constipation, diarrhea, mucositis, peripheral neuropathy (numbness, tingling), cognitive impairment, and pain. Each symptom is scored from 0 (absent) to 4 (severe/maximum). The survey will be administered at baseline and post-intervention to determine whether fasting interventions reduce the severity of chemotherapy-induced adverse effects. | Baseline and post-intervention (4 weeks for Groups 1-3; 2 weeks for Group 4) |
| Change in Blood Ketone Body Levels | Ketone bodies (primarily beta-hydroxybutyrate) are produced during fasting and ketogenic diet interventions and may mediate anti-tumor effects through metabolic and signaling mechanisms. Blood ketone levels will be measured using a ketone meter with test strips at baseline and at regular intervals during the intervention period. For Groups 2 and 3, measurements will be taken weekly during the 30-day intervention. For Group 4, measurements will be taken at days 3, 7, and 14 of the water-only fast. Ketone levels will be correlated with other outcome measures to explore the relationship between nutritional ketosis and therapeutic effects. | Baseline and during intervention: weekly for Groups 2-3 (30 days); days 3, 7, and 14 for Group 4 |
| 30442235 | Background | Sundaram S, Yan L. Time-restricted feeding mitigates high-fat diet-enhanced mammary tumorigenesis in MMTV-PyMT mice. Nutr Res. 2018 Nov;59:72-79. doi: 10.1016/j.nutres.2018.07.014. Epub 2018 Jul 31. |
| 34433808 | Background | Tang X, Li G, Shi L, Su F, Qian M, Liu Z, Meng Y, Sun S, Li J, Liu B. Combined intermittent fasting and ERK inhibition enhance the anti-tumor effects of chemotherapy via the GSK3beta-SIRT7 axis. Nat Commun. 2021 Aug 25;12(1):5058. doi: 10.1038/s41467-021-25274-3. |
| 30601864 | Background | Wilhelmi de Toledo F, Grundler F, Bergouignan A, Drinda S, Michalsen A. Safety, health improvement and well-being during a 4 to 21-day fasting period in an observational study including 1422 subjects. PLoS One. 2019 Jan 2;14(1):e0209353. doi: 10.1371/journal.pone.0209353. eCollection 2019. |
| 32958269 | Background | Riedinger CJ, Kimball KJ, Kilgore LC, Bell CW, Heidel RE, Boone JD. Water only fasting and its effect on chemotherapy administration in gynecologic malignancies. Gynecol Oncol. 2020 Dec;159(3):799-803. doi: 10.1016/j.ygyno.2020.09.008. Epub 2020 Sep 18. |
| 30093470 | Background | Myers TR, Zittel M, Goldhamer AC. Follow-up of water-only fasting and an exclusively plant food diet in the management of stage IIIa, low-grade follicular lymphoma. BMJ Case Rep. 2018 Aug 9;2018:bcr2018225520. doi: 10.1136/bcr-2018-225520. No abstract available. |
| ID | Term |
|---|---|
| D001943 | Breast Neoplasms |
| D000093763 | Intermittent Fasting |
| ID | Term |
|---|---|
| D009371 | Neoplasms by Site |
| D009369 | Neoplasms |
| D001941 | Breast Diseases |
| D012871 | Skin Diseases |
| D017437 | Skin and Connective Tissue Diseases |
| D005215 | Fasting |
| D005247 | Feeding Behavior |
| D001519 | Behavior |
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| ID | Term |
|---|---|
| D002985 | Clinical Protocols |
| D055423 | Diet, Ketogenic |
| ID | Term |
|---|---|
| D013812 | Therapeutics |
| D016020 | Epidemiologic Study Characteristics |
| D017531 | Health Care Evaluation Mechanisms |
| D011787 | Quality of Health Care |
| D017530 | Health Care Quality, Access, and Evaluation |
| D050528 | Diet, Carbohydrate-Restricted |
| D004035 | Diet Therapy |
| D044623 | Nutrition Therapy |
| D004032 | Diet |
| D009747 | Nutritional Physiological Phenomena |
| D000066888 | Diet, Food, and Nutrition |
| D010829 | Physiological Phenomena |
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