Low‐Dose Fisetin Supplementation and Its Association With Chronic Inflammation in Middle‐Aged and Older Adults: Study Protocol for a Triple‐Blind, Randomised, Placebo‐Controlled Trial

ABSTRACT

Systemic chronic inflammation contributes to the development of many age‐related chronic diseases and represents a promising target for interventions aiming at promoting healthy aging. Fisetin is a naturally occurring flavonoid with anti‐inflammatory, antioxidant and senolytic effects in vitro and in animal models, but evidence from randomised controlled trials in humans is limited. This study is a triple‐blind, randomised, placebo‐controlled trial designed to investigate the association between daily fisetin supplementation and changes in plasma soluble urokinase plasminogen activator receptor (suPAR) levels, as well as the occurrence and severity of adverse events, in generally healthy middle‐aged and older adults, with exploratory evaluation of additional biomarkers and functional outcomes. Participants aged ≥ 50 will be randomised to receive 100 mg of oral fisetin or placebo once daily for 7 weeks. The primary outcome is the between‐group difference in the change in plasma levels of suPAR from Baseline to Week 7. The secondary outcome is the between‐group difference in the occurrence and severity of adverse events during the study period. In addition, exploratory outcomes will evaluate the association of fisetin supplementation with other soluble and cellular biomarkers of inflammation, aging and cellular senescence, as well as measures of frailty, physical and cognitive function and subjective health.

Plain Language Summary

Fisetin is a natural compound found in fruits and vegetables that has shown anti‐inflammatory effects in laboratory studies. Fisetin is available as a nutritional supplement, but evidence of its effects in humans is limited. This study is designed to investigate whether middle‐aged and older adults who are randomly assigned to receive daily low‐dose fisetin supplementation for 7 weeks show changes in blood markers of inflammation and aging compared with participants receiving a placebo. The study will also evaluate side effects related to fisetin supplementation. The results will provide new knowledge about the safety and potential effects of fisetin supplementation.

1. Introduction and Background

Despite increases in life expectancy, most people spend the final decades of their life with age‐related chronic diseases or disabilities that diminish quality of life [1], resulting in a growing gap between lifespan (the number of years lived) and healthspan (the number of years lived in good health). This gap currently averages 9.1 years globally and is projected to increase by approximately 22% by the year 2100 [1, 2]. This has prompted growing public interest and research efforts toward preventive health strategies aimed at mitigating age‐related decline and promoting healthy aging. Under the premise of the geroscience hypothesis, targeting the fundamental mechanisms of aging offers a promising, broad‐reaching strategy to extend healthspan [3, 4], in contrast to the current approach of treating established diseases in isolation, which often leads to polypharmacy [5, 6], fragmented care and frequent visits to general practitioners and specialists [6, 7].

Among the hallmarks of aging, systemic chronic inflammation has emerged as a central driver of chronic disease development and therefore a key target for healthy aging [8]. This persistent, low‐grade, inflammatory status is driven, in part, by the accumulation of senescent cells and their senescence‐associated secretory phenotype (SASP) [8, 9]. Fisetin is a naturally occurring flavonoid polyphenol found in various fruits and vegetables, including strawberries, apples, persimmons, cucumbers, grapes, onions and tomatoes [10, 11], and has attracted attention for its potential to mitigate these age‐related processes. Fisetin has a wide range of beneficial effects in vitro, including anti‐inflammatory [12, 13, 14] and antioxidant [15, 16, 17] activities, and was recently shown to exhibit potent senolytic effects at high doses, extending healthspan and lifespan in animal models by selectively leading to the elimination of senescent cells and reducing SASP levels [18, 19, 20, 21, 22]. Fisetin is highly lipophilic and can therefore easily penetrate cell membranes to exert its effects [17, 23]; however, it displays limited bioavailability in vivo [24, 25, 26, 27, 28]. Following oral administration, fisetin is absorbed in the intestine and rapidly metabolised in the liver through sulfation, glucuronidation and methylation, resulting in low levels of the parent compound and high levels of metabolites in the circulation [24, 25, 26, 27, 28]. Nevertheless, both fisetin and its metabolites have been detected in multiple organs in preclinical models, including the liver, kidneys and brain, suggesting the ability to penetrate tissues [26, 29].

Low‐dose fisetin supplementation (100–200 mg daily) has been reported to reduce inflammatory biomarker levels in colorectal cancer patients undergoing chemotherapy [30], in patients with acute ischemic stroke [31], and to lower pro‐inflammatory adipokines in obese men [32]. It has also been associated with lower expression of SASP factors and a reduced proportion of senescent immune cells in a noncontrolled study of healthy individuals who self‐supplemented with fisetin [33]. However, robust evidence supporting its benefits in the general population remains limited. Although fisetin exhibits a favourable safety profile, in vitro studies indicate dose‐dependent inhibition of cytochrome P450 (CYP) enzymes, particularly CYP3A4 [34, 35], which may increase the risk of elevated plasma levels and accumulation of co‐medications metabolised by CYP3A4 when taken at high doses, or continuously for prolonged periods. These observations warrant the need for well‐controlled studies to evaluate the safety and anti‐inflammatory potential of fisetin supplementation in the general population.

However, reliably assessing chronic inflammation remains challenging, as traditionally used biomarkers, such as cytokines and acute phase reactants, can be drastically affected by transient events and may not accurately capture long‐term inflammatory status or chronic risk [36]. suPAR, the soluble form of the urokinase plasminogen activator receptor (uPAR), is an emerging biomarker of chronic inflammation, exhibiting a strong association with age and broad, nonspecific associations with multiple major chronic diseases and all‐cause mortality [37, 38, 39, 40, 41]. suPAR levels are relatively stable and largely unaffected by short‐term stressors [42, 43], but can be modified by lifestyle changes such as smoking and diet [44, 45], as well as therapeutic interventions in treatment of disease [36]. uPAR is both expressed on the surface of senescent cells and released as suPAR as part of the SASP [9, 46], and recent evidence suggests it may play a causal role in renal and cardiovascular diseases [47, 48]. Thus, suPAR has high potential as a biologically relevant measure of systemic chronic inflammation and as a responsive outcome measure for interventions targeting aging biology, in particular chronic inflammation and cellular senescence.

2. Objectives

The primary objective of this study is to assess whether daily low‐dose (100 mg) oral fisetin administered over 7 weeks is associated with changes in levels of biomarkers of systemic chronic inflammation, compared with placebo, in relatively healthy middle‐aged and older adults. Chronic inflammation will be measured by the change in plasma suPAR levels. The secondary objective is to evaluate the safety and tolerability of daily fisetin supplementation by monitoring the type and frequency of adverse events (AEs) reported during the study period. Furthermore, exploratory objectives will assess overall health benefits of daily fisetin supplementation, including associations with additional inflammation and aging‐related biomarkers, physiological, physical and cognitive function, as well as quality of life.

3. Study Design, Participants, Intervention and Outcomes

3.1. Setting, Study Design and Participant Timeline

The study is a single centre, investigator‐initiated, two‐arm, triple‐blind, randomised, placebo‐controlled trial (Figure 1) conducted at the Department of Clinical Research, Copenhagen University Hospital Amager and Hvidovre, Hvidovre (hereafter ‘Hvidovre Hospital’).

FIGURE 1.

Study design. Volunteers expressing interest in participating in the study will be screened and provided with oral and written information. After written informed consent is obtained, participants will be randomized to receive one capsule fisetin or placebo once daily for 7 weeks (50 capsules in total). Data and blood samples will be collected during in‐person study visits at baseline, Week 3, and Week 7. Adverse events and compliance data will be collected via telephone follow‐ups at Week 1 and through online questionnaires at Weeks 2 and 5. Figure created using BioRender.

Middle‐aged and older volunteers recruited from the general population will be randomised 1:1 in a parallel group design between the two treatment arms to receive either:

• One capsule (100 mg) fisetin once daily for 7 weeks (intervention group) or

• One capsule placebo once daily for 7 weeks (control group).

The study will be conducted over a period of 7 weeks and includes three in‐person visits: Baseline (Week 0), Week 3 and Week 7 during which outcome measurements will be performed (Figure 1). In addition, safety monitoring and treatment compliance will be assessed by phone interview at Week 1 and via self‐administered online questionnaires at Weeks 2 and 5. Follow‐up visits will be scheduled as close as possible to the planned timepoints for Week 3, 21 days after Baseline, with an acceptable range of −7 days to +4 days, and for Week 7, 49 days after Baseline, with an acceptable range of ± 5 days. To promote participant retention, visits will be scheduled to accommodate participant's availability and participants will receive reminders prior to each visit and completion of online questionnaire.

3.2. Eligibility Criteria

Inclusion and exclusion criteria for participation in the study are listed in Table 1. These criteria are designed to select a study population representative of relatively healthy middle‐aged and older adults for whom preventive interventions may be relevant.

TABLE 1.

Inclusion and exclusion criteria.

Inclusion criteria

Middle‐aged or older adult (≥ 50 years) Able to cooperate cognitively Able to read and understand Danish

Exclusion criteria

Inability or unwillingness to take oral supplements Chronic or recent (within 30 days) use of other supplements with potential antiaging effects (Table S1A) Chronic or recent (within 30 days) treatment with medications having potential antiaging effects (e.g., metformin, rapamycin, semaglutide; Table S1B) Chronic or recent (within 30 days) treatment with anti‐inflammatory medications Chronic or recent (within 30 days) treatment with medications that can interact negatively with fisetin listed in Table S1B (see further description below) Recent (within 14 days) vaccination Treatment with another investigational drug or other intervention with putative antiaging or anti‐inflammatory effects within 6 months a Active cancer or current cancer treatment Unstable or uncontrolled major disorders, for example, cardiovascular, renal, endocrine, immunological, hepatic disorder or cancer, requiring regular monitoring at the hospital Planned medical and surgical procedures during the study period Known hypersensitivity or allergy to fisetin or excipients in the placebo capsules Presence of any condition that, in the opinion of the investigator or study physicians, would place the participant at risk or preclude successful completion of the study

^a This criterion was changed from “Treatment with another investigational drug or other intervention within 1 year” in protocol amendment #1 (protocol version 4, 25.09.2025).

The medications listed in Table S1B may lead to exclusion, based on indication and dose, due to possible CYP interactions involving CYP3A4, CYP2D6, CYP2C9, or CYP2C8, which may have serious consequences due to treatment failure or overdose, particularly in cases involving HIV, fungal infections, severe psychiatric disorders, use of drugs with a narrow therapeutic index, or severe arrhythmic diseases. Additionally, some medications may introduce a potential bias with study outcomes or pose overall safety concerns for the participants. In addition to participants' self‐reported medication use, all medications will be verified through the shared medication records (FMK) by a study pharmacist or study physician after informed consent is obtained. FMK is a central database covering all Danish citizens and contains information on all medications prescribed and dispensed at primary care pharmacies during the previous two years [49].

A participant may decide to withdraw from the study or may be excluded from the study by the investigator before completing all the study‐related procedures for a variety of reasons, including participant safety concerns, failure of the participant to adhere to protocol requirements, incident disease or disease progression (as listed in exclusion criteria), or initiation of supplements or medications listed in Table S1 during the study period.

3.3. Recruitment

Participants will be recruited through posters at Hvidovre Hospital and a Danish online platform where they can register their interest in participating in clinical studies. To promote enrolment of participants, links to the study page on the online platform will be posted on social media. Volunteers who provide their contact details will be sent a link to a REDCap screening tool to complete a brief checklist regarding the use of commonly used medications and nutritional supplements with putative antiaging effects. Participants who indicate use of any medications or supplements listed in the exclusion criteria are automatically notified via REDCap that they are ineligible for participation. Volunteers who are not excluded based on this screening tool will be subsequently contacted by study personnel and will undergo a further preliminary screening by telephone, followed by an oral information session (can be conducted as an online video call). A reflection period of at least 24 h will be offered before informed consent is collected. After informed consent is obtained, the potential participants will be screened for prescribed medications and relevant diagnoses through review of their electronic medical records. Participants who meet the eligibility criteria will be subsequently enrolled in the study.

3.4. Intervention

To ensure product quality, fisetin and matching placebo capsules used in this study were contract‐manufactured and packaged by MoleQlar GmbH (Berlin, Germany) under EU Regulation (EC) No 178/2002, IFS Food Standard, HACCP (Hazard Analysis and Critical Control Point) standards. Fisetin capsules contain 100 mg fisetin (Novusetin, Bioriginal) and microcrystalline cellulose as filler. Placebo capsules contain microcrystalline cellulose and Exberry colouring Shade Lemon Yellow to match the appearance of fisetin. Both fisetin and placebo powders are encapsulated in size 1 white vegan hydroxypropyl methylcellulose capsules identical in shape, size and colour to ensure blinding of participants and study personnel. Capsules are packed in bottles of 25 and stored at room temperature in accordance with the manufacturer's instructions. Certificates of analysis provided by the manufacturer confirm identity, strength and purity of fisetin and placebo capsules. Stability of fisetin in the capsules will be monitored from release through 24 months to ensure product integrity throughout the study period.

Participants will receive one orally administered capsule containing fisetin (100 mg) or placebo once daily for 7 weeks, for a total of 50 capsules. At the Baseline visit, each participant will receive one bottle containing 25 capsules of the allocated treatment, and the first dose will be administered on‐site by study personnel. Thereafter, the participants will self‐administer one capsule daily. A second bottle containing 25 capsules will be provided at the Week 3 visit to allow continued daily dosing until the Week 7 visit. Participants will be instructed to ingest the capsule with water, either before or during a meal, and will be encouraged to use reminders to support compliance with the intervention. They will also be instructed to return the bottles, including any remaining capsules, at the Week 3 and Week 7 visits for assessment of compliance by capsule count. Treatment compliance will also be assessed using a questionnaire at each follow‐up assessment (Weeks 1, 2, 3, 5, 7).

3.5. Outcomes

The primary outcome measure is the between‐group difference in the change in plasma suPAR levels from Baseline to Week 7. Levels of suPAR will be measured in EDTA plasma samples using the suPARnostic (Virogates A/S, Denmark) enzyme‐linked immunosorbent assay (ELISA). The secondary outcome is the safety of the intervention, assessed as the between‐group difference in the frequency and severity of AEs during the study period, with particular attention to gastrointestinal symptoms, fatigue, headaches and dizziness.

Additional exploratory outcome measures, including measurement variables and timeframe for assessment, are presented in Table 2.

TABLE 2.

Exploratory outcomes.

Outcome	Description	Timepoint
Blood biomarkers of inflammation, biological aging, and cellular senescence
Soluble inflammation and SASP biomarkers	The change in plasma levels of SASP factors and inflammation markers (e.g., cytokines, chemokines, proteases, growth factors, cell free mitochondrial DNA [cf‐mtDNA]).	Baseline, Week 3, Week 7
Cellular senescence biomarkers	The change in expression levels of senescence‐associated measures, including marker expression (e.g., p16INK4a, p21CIP1/WAF1, uPAR) in PBMCs and T cells, as well as morphological features in blood smears.	Baseline, Week 7
Soluble aging biomarkers	The change in plasma levels of aging markers (e.g., α‐klotho, growth differentiation factor 15, fibroblast growth factor 21).	Baseline, Week 3, Week 7
Cellular aging biomarkers	The change in DNA methylation patterns and epigenetic age in buffy coats.	Baseline, Week 3, Week 7
Immune function biomarkers	The change in PBMC subset distribution and activation.	Baseline, Week 7
Oxidative stress biomarkers	The change in plasma levels of lactate, creatine kinase, pyruvic acid, and creatine.	Baseline, Week 3, Week 7
Phenotypic age	The change in biological age computed using the PhenoAge algorithm [50].	Baseline, Week 7
Functional parameters and markers of organ function
Frailty	The change in frailty status calculated as Frailty Index OutREF (FI‐OutRef; i.e., the number of routine biomarkers outside reference range [51]).	Baseline, Week 7
Kidney function	The change in plasma levels of creatinine and/or cystatin C.	Baseline, Week 3, Week 7
Physical function	The change in physical function assessed by 4‐m gait speed, hand grip strength, chair stand test, and balance.	Baseline, Week 7
Cognitive function	The change in cognitive function assessed using the MoCA test and the Digit Symbol Substitution Test.	Baseline, Week 7
Quality of life	The change in quality of life assessed using the EuroQol‐5D‐5L [52, 53].	Baseline, Week 7
Self‐rated health	The change in self‐rated health.	Baseline, Week 7
Self‐reported physiological age	The change in self‐reported physiological age.	Baseline, Week 7

Abbreviations: MoCA, Montreal Cognitive Assessment; PBMCs, peripheral blood mononuclear cells; SASP, senescence‐associated secretory phenotype; uPAR, urokinase plasminogen activator receptor.

3.6. Harms

In addition to the study outcomes, AEs and adverse reactions (ARs) will be monitored and documented throughout the study period and documented in the respective participants' case report forms (CRFs) and in the annual and final safety reports. Participants will be asked about the occurrence and severity of common or expected side effects of fisetin (safety outcomes described above) at each follow‐up timepoint, including when the symptoms started, how long they lasted and whether they occurred before or after intake of the pill. In addition, participants will be encouraged to report spontaneously any other AE/ARs or unintended effects. Participants experiencing AE/ARs will be monitored until the event is stabilised or resolved, and the investigator will determine whether to discontinue the intervention or withdraw the participant from the study. All AE/ARs will be evaluated by a medical doctor for severity, duration, outcome, expectedness and potential relation to the intervention. Serious adverse events (SAEs) and reactions (SARs) will be reported to the Scientific Ethics Committee of the Capital Region of Denmark in accordance with reporting requirements.

3.7. Sample Size Determination

The study will be conducted with a fixed sample size determined by feasibility constraints. Based on the available stability data for the active ingredient used in the investigational product (expiration 24 months after manufacturing) and anticipated recruitment capacity, a total sample size of 120 participants, with equal allocation to intervention and control groups, represents the maximum number of participants expected to be enrolled and treated within the available period.

At present, no data on the change in suPAR levels after fisetin supplementation in this population and over a similar time frame were available from previous studies. Therefore, a power estimation was performed to determine the minimum between‐group difference in change that could be detected with the planned sample size. Longitudinal data from the FAM‐CPH cohort conducted by our group [54] were used for the power calculation, despite this cohort being younger than the target population of the present study. Data from healthy individuals aged 20–35 years, measured at two timepoints 4 weeks apart without intervention, were multiplied by two to approximate changes over a period similar to the present study (7 weeks), suggesting that the change in suPAR levels is on average +5% with a standard deviation of 22%. The power calculation thus indicates that 60 participants per group provides the study with 84.2% power to detect a 12% difference between groups in the change in suPAR levels from Baseline to Week 7 at a significance level of 0.05.

Any randomised participants who have withdrawn or have been excluded from the study may be replaced to achieve the target sample size of 120 treated participants, provided study completion is possible within the available period.

3.8. Randomisation

A statistician will prepare a computer‐generated block‐randomisation schedule using the statistical software R [55] to allocate the participant to either of two treatment codes (A or B) in a 1:1 ratio. Randomisation will be performed in blocks of four and stratified by sex to ensure a balanced distribution of men and women across treatment groups, as sex differences may influence biomarker levels and response to the intervention. To maintain blinding, the treatment codes A/B will be linked to the actual study product (fisetin/placebo) by an independent, unblinded randomisation manager not involved in the study. Password‐protected files for each participant ID will be stored in a secure folder accessible to the randomisation manager and the study physicians. The passwords, full randomisation list and treatment code key will be stored in a separate secure folder accessible only to the randomisation manager. The study products will be labelled by the randomisation manager with only the participant identification number according to the randomisation schedule and delivered to the participants by blinded study personnel. All participants and study personnel involved in data collection and analysis, including laboratory analyses, will be blinded to intervention assignment for the entire study duration. In the event of a SAE/SAR or if knowledge of the treatment allocation is essential for further management of the participant, the study physician will be provided with the relevant password to unblind an individual participant. Unblinding for any other reason will be considered a protocol deviation.

4. Data Collection, Management and Analysis

4.1. Data Collection Methods

Data collection methods for the study's descriptive variables (including parameters that may influence or confound treatment effects, i.e., CYP3A4 activity, body composition, sex hormones levels and dietary habits) and outcomes are detailed in Table 3. A research biobank will be established for batch analysis of plasma, serum, buffy coats and peripheral blood mononuclear cells (PBMCs). Serum, EDTA plasma and buffy coats will be collected at the Baseline, Week 3 and Week 7 visits, isolated by centrifugation and stored at −80°C. At the Baseline and Week 7 visits, PBMCs will be isolated from EDTA‐treated whole blood by density gradient centrifugation under sterile conditions, then aliquoted and stored at −150°C. T cells will be isolated from an aliquot of PBMCs using negative magnetic selection. Both the isolated T cells and a separate aliquot of bulk PBMCs will be preserved in TRIzol and stored at −80°C for qPCR analyses. In addition, EDTA‐treated whole blood will be used to prepare Giemsa‐stained blood smears for morphological assessment of cellular senescence. Samples in the research biobank will be used for assessment of the outcomes described in Table 3 and may also be used for additional analyses, including proteomics, metabolomics and transcriptomics, or future biomarker studies.

TABLE 3.

Assessment methods and timepoints for study outcomes.

Variable	Method	Timepoint
Descriptive variables
Participant characteristics	Demography (i.e., marital status, living conditions, education, and occupational status) and lifestyle (smoking, alcohol and substance use, and physical activity) data collected via questionnaires, as well as anthropometric (height and weight) and clinical (blood pressure and pulse a ) data collected via physical examinations. Data on hospitalizations, diagnoses, and prescribed medication obtained from the participants' electronic medical records and the shared medication record (FMK).	Baseline, Week 7
Dietary habits	Dietary quality assessed using a 23‐item food frequency questionnaire [56]. Usual intake of fisetin through the diet assessed using a questionnaire about consumption of fisetin‐rich foods [10, 11]. Current use of other nutritional supplements assessed using a questionnaire a . Nutritional status assessed using the Mini Nutritional Assessment – Short Form (MNA‐SF) [57].	Baseline, Week 7
Cytochrome P450 3A4 (CYP3A4) activity	Assessed using the endogenous marker 4β‐hydroxycholesterol measured in plasma samples with LC–MS/MS to evaluate fisetin's inhibitory effects on CYP3A4 activity.	Baseline, Week 3, Week 7
Body composition a	Assessed by bioimpedance analysis (InBody S10 Body Water Analyser InBody, Cerritos, California, USA) to examine whether body composition influences study outcomes.	Baseline, Week 7
Sex hormones	Levels of oestrogen, testosterone, or other relevant hormones will be measured to explore their influence on study outcomes.	Baseline, Week 7
Compliance	Assessed by capsule count and participant questionnaires.	Week 1, 2, 3, 5, 7
Health events and medicine use	Assessed via questionnaires on acute illness, vaccinations, and changes in medications.	Baseline, Week 1, 2, 3, 5, 7
Primary outcome
suPAR	Levels of suPAR measured in EDTA‐treated plasma samples using an enzyme linked immunosorbent assay (ELISA).	Baseline, Week 7
Secondary outcome
Safety	Type and frequency of symptoms and adverse events assessed using a questionnaire which evaluates the presence and severity (Visual Analog Scale [VAS]‐scale ranging from 1 to 10) of commonly reported side‐effects of fisetin [24, 30, 58]: gastrointestinal symptoms, fatigue, dizziness and headaches/migraines, as well as any other symptoms spontaneously reported by participants.	Week 1, 2, 3, 5, 7
Exploratory outcomes
Blood‐based biomarkers
Soluble inflammation and SASP biomarkers	Plasma or serum levels of e.g., suPAR, IL‐6, IL‐8, IL‐10, IL‐18 and MMPs measured using commercially available immunoassays such as ELISA, Ella or Luminex methods, as well as cf‐mtDNA measured using qPCR.	Baseline, Week 3 b , Week 7
Cellular senescence biomarkers	Flow cytometry and qPCR analysis of e.g., p16INK4a, p21CIP1/WAF1 and uPAR expression in PBMCs and T cells. Microscopy analysis of Giemsa‐stained whole blood smears to evaluate cellular morphology [59].	Baseline, Week 7
Soluble aging biomarkers	Plasma or serum levels of e.g., GDF15, FGF21 and α‐klotho measured using commercially available immunoassays.	Baseline, Week 3 b , Week 7
Cellular aging biomarkers	DNA methylation in buffy coats analysed using a validated array.	Baseline, Week 7
Immune function biomarkers	Flow cytometry analysis of subset distribution, functionality and immunosenescence markers in PBMCs.	Baseline, Week 7
Oxidative stress biomarkers	Plasma or serum levels of e.g., lactate, creatine kinase, pyruvic acid and creatine measured using commercially available assays.	Baseline, Week 7
Phenotypic age	Calculated from blood biochemistry using the PhenoAge algorithm [50].	Baseline, Week 3, Week 7
Functional parameters and markers of organ function
Frailty	FI‐OutRef, a lab‐based frailty index derived from the number of routine clinical biochemistry tests outside of their normal reference range, reflecting cumulative physiological dysregulation [51, 60].	Baseline, Week 7
Kidney function	Assessed by levels of creatinine and cystatin C measured using routine clinical assays.	Baseline, Week 3, Week 7
Physical function	Assessed using the 4‐m gait speed test (usual gait speed) [61], 30‐s chair‐stand test [62], handgrip strength measured using a hand dynamometer [63], and balance evaluated with the Short Physical Performance Battery balance test [64, 65].	Baseline, Week 7
Cognitive function	Assessed using the Montreal Cognitive Assessment (MoCA) [66] and Digit Symbol Substitution Test (DSST) [67, 68].	Baseline, Week 7
Quality of life	Assessed using the EuroQol‐5D‐5L [52, 53] (Reg. ID: 78228).	Baseline, Week 7
Self‐rated health	Assessed as the answer to the question: “How do you think your health is, all in all?,” with the following possible answers: “excellent,” “very good,” “good,” “fair” or “bad.”	Baseline, Week 7
Self‐reported physiological age	Assessed as the answer to the question: “Chronological (number of years since birth) and physiological age are two different things, how old do you feel at present?”.	Baseline, Week 7

Abbreviations: FI‐OutRef, Frailty Index‐OutRef; FGF21, fibroblast growth factor 21; GDF15, growth differentiation factor 15; HDL, high‐density lipoprotein; IL, interleukin; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LDL, low‐density lipoprotein; MMPs, matrix metalloproteinases; PBMC, peripheral blood mononuclear cells; suPAR, soluble urokinase plasminogen activator receptor.

^a These outcomes were added to the protocol in amendment #1 (version 4, 25.09.2025).

^b Selected biomarkers only.

4.2. Data Management

The primary data collection tool will be an electronic case report form (eCRF) using the REDCap platform (Research Electronic Data Capture, Vanderbilt University, Nashville United States). Data will be entered directly in the eCRF, and data integrity and completeness will be ensured through a variety of mechanisms such as range checks, checks of valid values, checks for data completion and locking of completed data entry fields. Although paper‐based CRFs will be used where necessary, for example, for cognitive testing sheets, all data will be immediately entered into the eCRF. Data from experimental laboratory analyses will be exported in Excel files and saved on a secure drive.

4.3. Statistical Methods

Data will be analysed in R [55]. For descriptive statistics, continuous variables will be presented using mean and standard deviation, or medians and interquartile range for non‐normally distributed variables and counts and percentages for categorical variables. Statistical significance will be defined as a two‐tailed p‐value < 0.05 and will be corrected using Bonferroni correction whenever multiple comparisons are performed.

For analysis of the primary outcome, data from all participants who completed the study as well as those with withdrew will be included. Analyses will use available data, and data assumed to be missing at random will be handled using multiple imputation. For data assumed to be missing not at random, for example, where withdrawal is considered likely to be related to AEs, a worst‐case sensitivity analysis will be performed. In addition, a sensitivity analysis including only participants with complete data will be performed. To assess the extent of any potential bias, baseline characteristics of participants who withdrew or are lost to follow‐up will be compared with that of those who completed the study. An additional sensitivity analysis will be conducted using data from participants who attend all three in‐person visits (Baseline, Week 3 and Week 7) and demonstrate high compliance, defined as consumption of at least 90% of capsules, with a maximum of no more than one missed capsule per week. The primary analysis will compare the change in plasma suPAR levels from Baseline to Week 7 between intervention and placebo groups. Adjusted analyses will be performed using linear regression models, with appropriate data transformations or bootstrap methods applied if model assumptions are not met. Adjusted analyses will include baseline levels of the respective outcome measures in the models, as well as other variables identified as relevant confounders. Unadjusted comparison of the change will be analysed using an independent two‐sample t‐test, or the Wilcoxon rank‐sum test if normality assumptions are not met.

For analyses of the secondary outcome, frequency tables will be used to summarise the occurrence of AEs/ARs during the study period by treatment group. Comparisons of the percentage of individuals with AEs/ARs and total number of AEs/ARs between groups will be done using chi‐square or Fisher's exact test. Comparisons will be done for each type of AE/AR separately. Finally, comparisons of the severity of AEs/ARs will be done using Student's t‐test or Wilcoxon rank‐sum test.

Exploratory outcomes, including group differences in biomarker changes from Baseline to Week 3 and to Week 7, will be analysed similarly using t‐tests or Wilcoxon rank‐sum tests, and chi‐square or Fisher's exact test for categorical variables. Adjusted analyses will use linear or logistic regression models as appropriate. Repeated measures will be analysed using paired t‐tests or linear mixed‐models. Relationships among exploratory variables will also be explored using correlations and regression models.

5. Ethics and Monitoring

5.1. Research Ethics Approval

The trial will be conducted in accordance with the Declaration of Helsinki and has been approved by the Scientific Ethics Committee of the Capital Region of Denmark (approval # H‐25043857). Given the low risk of the intervention, no data monitoring committee was established for this study. Ongoing safety evaluation will be conducted by the study personnel and investigators. A revision of the treatment regimen or a general interruption of the study may be relevant if the risk of conducting the study is too great, that is, in the occurrence of one SAR attributed to the investigational product in this study, or in other trials using similar doses.

5.2. Protocol Amendments

One protocol amendment (amendment #1) has been reviewed and approved by the Scientific Ethics Committee for the Capital Region of Denmark (protocol version 4, 25.09.2025). The amendment includes the following changes: The exclusion criteria regarding treatment with other investigational drugs or interventions within the past year were updated to specify that the treatment or intervention must have a potential effect on inflammation or aging biology, and the timeframe has been reduced to 6 months; a questionnaire was added to capture information on the participants' use of other nutritional supplements throughout the study period; collection of data on pulse was added; bioimpedance measurement has been added to evaluate effect of body composition on the intervention; the frailty index FI‐OutRef [51] at Week 3 has been removed as it was not expected to contribute significant information and blood sample volumes have been corrected accordingly; the recruitment material has been revised to include additional details on exclusion criteria and include a QR code redirecting to a REDCap screening tool—these changes aim to improve participant experience and facilitate contact while reducing participation requests from potential participants not meeting eligibility criteria.

Any future substantial amendments will be submitted for approval before implementation.

5.3. Consent

All participants will receive written and oral information and must provide written informed consent prior to enrolment, including consent for the collection and storage of blood samples in a research biobank for current and future analyses related to the study objectives. Written informed consent will be obtained by trained study personnel, and participants will be informed that they may choose to withdraw from the study at any time and request the destruction of any biological materials collected as part of the study.

5.4. Ancillary and Post‐Trial Care

Participants will receive necessary medical care for any AEs or complications related to trial participation. No post‐trial care will be offered, except for ongoing management of eventual trial‐related AEs or complications. Participants are covered under the Danish Patient Compensation Act.

5.5. Confidentiality, Access to Data and Dissemination Policy

The study is registered in the Data Registry for the Capital Region of Denmark (via the legal repository Privacy; registration #p‐2025‐19 591). Participant information will be kept confidential and managed in compliance with the General Data Protection Regulation (GDPR) and Danish Law. Each participant will be assigned a pseudo‐anonymised ID used for data collection and analysis. The identification key linking ID numbers to personal information will be stored in REDCap, as well as on a secure drive with limited access. All study‐related information about the participants will be registered in eCRFs, whereas paper‐based CRFs will be securely stored in locked cabinets. Biobanked samples will be pseudo‐anonymised.

The results of the study, whether positive, negative, or inconclusive will be disseminated through scientific articles in international peer‐reviewed journals in accordance with the CONSORT guidelines [69] and presented at national and international conferences. There will be no personally identifiable data in the publications. The results of the trial for the primary and secondary outcomes will be published and posted on clinicaltrials.gov within 12 months after completed data collection.

6. Discussion

Currently, most of the evidence regarding the anti‐inflammatory and antioxidant effects of fisetin and other nutritional supplements stems from in vitro experiments and animal models [18, 19, 70, 71, 72], with only extremely limited evidence from studies in humans. In view of the growing popularity and use of nutritional supplements, particularly among relatively healthy individuals and older adults [73, 74, 75], there is a need for robust evidence to support their purported benefits and efficacy and evaluate their safety in humans.

The quality and composition of commercially available supplements, including fisetin, can vary substantially due to differences in formulation, manufacturing procedures and less stringent regulatory oversight compared with pharmaceutical products produced under Good Manufacturing Practice (GMP) conditions or with certificates of analysis. This variability can influence the quality, dosing and purity of supplements, potentially affecting both their efficacy and safety [76, 77]. The investigational product used in this trial was manufactured specifically for this study with certificates of analysis to ensure standardised composition and quality. A key strength of this study is the triple‐blind, randomised, placebo‐controlled design, which minimises bias and addresses limitations of previous studies [33]. Another strength of the study is the exclusion of individuals using other supplements or drugs with suspected potential ‘antiaging’ effects, which allows a clearer interpretation of the effects of the investigational product tested in this study. In addition to the selection of a robust and reliable measure of chronic inflammation as the primary outcome, the study also includes a broad range of exploratory outcomes spanning major components of aging, including inflammatory, senescence and immunosenescence markers, but also physical and cognitive function, as well as subjective self‐reported health measures. These will offer a comprehensive evaluation of fisetin's potential benefits. Finally, the inclusion of relatively healthy middle‐aged and older adults representing a segment of the population likely to self‐administer supplements or lifestyle interventions for health optimisation, as well as the broad inclusion criteria and few exclusion criteria, increases the relevance and generalisability of the study. This is also a population who might benefit from preventive interventions targeting early, preclinical signs of aging, before the onset of chronic disease and multimorbidity.

The short duration of the intervention was selected to promote compliance and low dropout rate; however, this limits the ability to evaluate long‐term effects and safety of the investigational product. Given the low oral bioavailability of fisetin [24], the dose used in the study may limit its potential efficacy. Although formulations with enhanced bioavailability are available [24], a simple formulation was selected to mirror the most common commercially available fisetin supplements. Several ongoing clinical trials are evaluating the pharmacokinetics, safety and efficacy of fisetin using high‐dose intermittent administration (typically 20 mg/kg for two consecutive days) for various disease indications [78, 79]. These high and transient doses aim to achieve high tissue penetration and thereby senolytic effects. Due to the pleiotropic properties of fisetin, the continuous low‐dose approach used in our study may lead to different biological effects such as lowering chronic inflammation via other mechanisms including antioxidant activity. Fisetin may influence inflammatory and metabolic pathways as suggested by a recent 12‐week study of low‐dose (200 mg daily) fisetin supplementation in obese men, which resulted in reductions in body weight and circulating levels of MCP‐1 and asprosin [32]. In addition, synergistic effects on inflammation and metabolism were observed when fisetin was combined with aerobic training [32]. The use of suPAR as primary outcome will inform on its relevance and utility as a clinically relevant outcome in future trials targeting chronic inflammation and aging biology.

In conclusion, this trial will provide important new knowledge on potential anti‐inflammatory effect of low dose fisetin supplementation in relatively healthy middle‐aged and older individuals.

7. Administrative Information

This study protocol is reported in accordance with the SPIRIT guidelines [80, 81]. A structured summary of the trial design and methods is presented in Table S2. A summary of protocol versions and amendments is presented in Table S3. The study will be conducted in accordance with the Basic and Clinical Pharmacology and Toxicology policies for experimental and clinical studies [82], and for natural products [83].

Author Contributions

Conceptualisation: JT, MB, LFH, JON, MBH, OA, LJHR. Writing – initial draft: JT, MB, LFH, LJHR. Writing – review and editing: All authors. Final approval of manuscript: all authors.

Funding

The trial is financed by the Department of Clinical Research, Amager & Hvidovre Hospital. JT is supported by grants from the Capital Region of Denmark's Research Funds (Region Hovedstadens Forskningsfond). LJHR is supported by grants from the ROCKWOOL Foundation, DFF Inge Lehmann (grant ID 10.46540/3162‐00023B), Brødrene Hartmanns Fond, Beckett‐Fonden, and the Amager and Hvidovre Hospital Research grant. The funding sources have no influence on the study design, data collection, interpretation of data or writing and reporting of the results.

Conflicts of Interest

None of the investigators have personal financial interests in fisetin. OA is a named inventor on patents on suPAR as a prognostic biomarker; the patents are owned by Copenhagen University Hospital Amager and Hvidovre, Denmark, and licensed to ViroGates A/S, the company that produces the suPARnostic ELISAs. ViroGates A/S has not provided financial or other support to the study, and they have not been and will not be involved in any parts of the study. MBH is a member of the editorial board of Basic and Clinical Pharmacology and Toxicology but was not involved in the peer‐review or editorial handling of this manuscript, in accordance with the journal's policy. The remaining authors declare no conflicts of interest.

Supporting information

TABLE S1: List of supplements and medications that lead to exclusion from the study.

TABLE S2: Structure summary of the trial.

TABLE S3: Protocol versions and amendments.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.