Investigation of Short and Intermediate Term Effects of a Supplement Mix Designed to Target Ageing Mechanisms on Vascular Function in Healthy Middle-aged Participants (STAMINA Study)
Phase: NA
Conditions: Healthy Aging, Vascular Dilation
Sponsor: University of Surrey

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
 
 
FULL/LONG TITLE OF THE STUDY 
Investigation of short and intermediate term effects of a supplement mix designed to target ageing 
mechanisms on vascular function in healthy middle-aged participants (STAMINA Study) 
 
SHORT STUDY TITLE / ACRONYM 
Effects of a supplement to target ageing mechanisms on vascular function (STAMINA Study) 
 
PROTOCOL VERSION NUMBER AND DATE 
V5.0 23/02/2024 
 
NCT number  
NCT06145087 
 
 
 
 
Confidentiality Statement 
 
The Information contained in this document is the property of the University of Surrey and is 
provided to you in confidence as an investigator, potential investigator, or consultant for 
review by you, your staff and applicable regulators. It is understood that this information will 
not be disclosed to others without authorisation from the University of Surrey except to the 
extent necessary to obtain written consent from those persons who will take part in the study. 
 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
STUDY SUMMARY 
Study Title 
Investigation of short and intermediate term effects of a 
supplement mix designed to target ageing mechanisms on 
vascular function in healthy middle-aged participants 
(STAMINA Study) 
Internal ref. no. (or short title) 
STAMINA 
Planned Size of Sample (if applicable) 60 
Planned Study Period 
01/12/2022 – 01/11/2024 
 
 
FUNDING AND SUPPORT IN KIND (Optional) 
FUNDER(S) 
(Names and contact details of ALL organisations 
providing funding and/or support in kind for this 
study) 
FINANCIAL AND NON FINANCIAL SUPPORT 
GIVEN 
AgeLessSciences, a Public Benefit Corporation,  
Kris Verburgh, PhD CEO email: 
kris@novoslabs.com 
Charitable donation to Prof. Heiss plus provision 
of supplement and placebo 
 
INVESTIGATORS 
NAME 
Position 
Signature (optional) 
Prof. Christian Heiss 
Professor of Cardiovascular 
Medicine  
 
Dr. Danuta Sampson 
Visiting research fellow 
 
Mr. Abdullah Durrani 
PhD student 
 
Dr. Mariam Bapir 
PhD student/post-doctoral 
researcher 
 
Mr. Charles Piercy 
PhD student 
 
Ms. Kenza Sackho 
PhD student 
 
 
 
 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
STUDY PROTOCOL 
1. Abstract 
In our ageing society, extending the healthy lifespan is a major challenge and a healthy diet may play 
an import role in maintaining health throughout life. With increasing age cardiovascular function declines 
and large and small blood vessels change in various and complex ways and these changes may lead 
to many age-related diseases. On a molecular level, there are many mechanisms that are associated 
with ageing including cellular senescence, loss of proteostasis, altered cellular communication, genomic 
instability, epigenetic alterations, telomere shortening, deregulated nutrient sensing, stem cell 
exhaustion and protein crosslinking. Animal and human studies suggest that dietary supplements may 
be able to affect these mechanisms. What the effect of the NOVOS supplement is on cardiovascular 
functions is not known. 
The aim of the present study is to investigate the short and intermediate term effects of a supplement 
mix designed to target ageing mechanism on vascular function in healthy middle-aged subjects. 
(STAMINA Study) We hypothesise that the supplement will lead to acute and sustained changes in 
biomarkers of vascular function and health. 
We will recruit 60 healthy middle-aged people and randomly assign them to either daily intake of the 
NOVOS supplement (n=30) or placebo (n=30) for 6 months. The supplement (NOVOS) is a 
commercially available product and has 13 components. It was developed and is provided together with 
the placebo by AgeLess Sciences LLC, a Public Benefit Corporation. The study will require 2 visits by 
participants during which non-invasive vascular exams will be performed, venous blood taken and spot 
urine sample collected (Visit 1: 3 h, visit 2: 60 min). The primary endpoint is change in flow-mediated 
dilation, secondary endpoints are change in blood pressure, cholesterol, arterial stiffness, microvascular 
function cardiovascular risk SCORE and daily walking distance. Tertiary endpoints are changes in 
biomarkers of ageing as assessed in blood samples including DNA damage. In addition, we will assess 
anxiety, depressive feelings, happiness, well-being and the diet with several questionnaires. After 
consent, measurements will be taken on the first day before and 2 hours after ingestion of the first 
supplement or placebo. Participants will consume the supplement or placebo for 6 months and vascular 
exams and one blood draw will be repeated during the final visit. During the time, participants will receive 
2 phone calls to improve compliance. 
2. Background or rationale of the project   
In our ageing society, extending the healthy lifespan is a major challenge and a healthy diet may play 
an import role in maintaining health throughout life. We know that with increasing age cardiovascular 
function declines and that large and small blood vessels change in various and complex ways and that 
these changes are also associated with the development of many age-related diseases. While vascular 
ageing is a key determinant of healthy ageing overall, a reason for the complexity of vascular ageing is 
that there are many molecular mechanisms involved. Animal and human studies suggest that dietary 
supplements may be able to affect these mechanisms. 
According to the classical free radical theory of ageing, production of endogenous oxygen radicals 
results in cumulative damage to the genetic information (DNA) in cells that line the blood vessels, 
endothelial cells, which is not efficiently repaired. Accumulation of unrepaired DNA damage may then 
lead to accumulation of not properly functioning cells that cannot divide anymore (senescent cells) but 
cause inflammation (‘inflamageing’). More recently it is appreciated that ageing is more complex, and 
the hallmarks of ageing include genomic instability, telomere attrition, epigenetic alterations, loss of 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell 
exhaustion, and altered intercellular communication.1,2 
A large body of research indicates that many of these processes can be modulated by various 
substances, including natural ingredients, micro-nutrients, and substances present in the human body 
and of which concentrations decline during aging.2 For instance novel evidence suggests that molecules 
that are present in fruits and vegetables – polyphenols – can reduce the risk or slow down the progress 
of aging-related diseases such as cardiovascular disease,3 Alzheimer’s disease, macular degeneration, 
and cancer.4 Other substances with proven effects individually include alpha-ketoglutarate,5-9 
glucosamines, glycine,10-15 nicotinamide mononucleotide,16-18 theanine,19-21  magnesium22-24 and 
ascorbic acid. Overall the results for most of these isolated nature-derived and nutritional bioactives 
have shown both in humans and mice that vascular function and genetic stress response pathways can 
be improved in both young and old over hours to weeks.25,26 However, we do not know if consumed over 
longer periods of time or even throughout life, this would affect the gradual ageing of blood vessels over 
months and years. While these studies with individual components are promising, combining 
components that promise to act on different mechanisms implicated in the ageing process and have 
synergistic effects seems like a more effective way to target ageing than focusing on one. How this 
combined approach affects vascular function and health parameters is not known.  
The aim of the study is to assess aging molecular pathways and biomarkers while consuming a 
supplement with a combination of components to address multiple molecular pathways of aging 
simultaneously. 
 
3. Patient/Participant involvement 
We have discussed our vascular ageing research including development of monitoring devices, 
research into potential interventions including dietary supplements and planned studies in the Surrey 
Healthy Ageing Research Partnership (SHARP). Most of the (lay) participants have indicated that they 
would be keen to participate in a future study and provided contact information. We are planning 
another PPI event soon to explain and promote the study. 
4. Aims and objectives 
The aim of the present study is to investigate the short and intermediate term effects of a supplement 
mix designed to target ageing mechanism on vascular function in healthy middle-aged subjects 
(STAMINA Study). We hypothesise that the supplement will lead to acute and sustained increases in 
vascular function. 
5. Benefits of the study 
The benefits to the participants will include better understanding of their current stage of vascular health 
and biological vascular age. The comprehensive information on the participants vascular function and 
health is not generally available in the NHS and quite expensive, if at all available, in the private sector 
and includes endothelial function, arterial stiffness/age, cholesterol, glucose and inflammatory markers. 
In addition, it will provide information on how these progress over half a year. Half of the participants will 
benefit from being provided with high quality supplement mix worth each approximately £500. In terms 
of knowledge generation, it will provide first data on what the combined effect of a supplement mix will 
be on vascular biomarkers that are associated with vascular ageing. The data will help to evaluate the 
potential of the supplement mix to modulate vascular function and plan future studies. 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
To contribute to participant’s travel expenses and time we offer £50 after completion of the study. For 
this, we will collect bank details which will not be share outside the study team. The bank details do not 
need to be provided this if the participant opts out and prefers to not receive it.  
The expenses will be submitted by Mariam Bapir to visitorexpenses@surrey.ac.uk and bank transfers 
processed by Faculty Finance. The bank details (recipient's name, sort code, account number) will only 
be used to be filled into the expense form. As soon as visitorexpenses@surrey.ac.uk confirm the 
expense have been processed, the study files with bank details and emails/sources with the bank details 
will be deleted.  
 
6. Recruitment Methods 
Recruitment will be via word to mouth, posters/advertisements at the university, social media posts and 
PPI event organised together with the Surrey Healthy Ageing Research Partnership. See attached 
posters and social media posts. 
7. Adverse Publicity  
We would not expect any adverse publicity. Rather the opposite as this study will help to understand if 
and how supplements affect vascular ageing and health.   
8. Informed Consent and Withdrawal of Consent 
Potential participants will be contacted, or they will contact us by telephone or email in response to word 
to mouth, posters or social media posts. We will explain the study briefly and provide the participant 
information material and consent form by mail or email. On the first visit, we will provide further 
information and answer questions the participants may have. After written informed consent is obtained, 
the participants will be screened for inclusion and exclusion criteria. If they fit with the criteria, they will 
be included, and we will then perform baseline measurements. 
Inclusion criteria: 
• 
Generally healthy. (Note: those with stable conditions that do not interfere with the study 
objectives/procedures will be included at the PI discretion).  
• 
>40 years 
• 
BMI >20 kg/m2 
• 
Systolic blood pressure >=120 mmHg 
• 
Smartphone with step counter 
Exclusion criteria: 
• 
Symptoms of acute infection 
• 
Cardiac arrhythmias  
• 
Active malignancy 
• 
Clinical signs or symptoms of unstable cardiovascular disease (coronary artery disease, lower 
extremity artery disease, cerebrovascular disease). These include angina pectoris, dyspnoea, 
palpitation, syncope, claudication, active vasoactive medication.  
• 
Women who have been pregnant in the last three months, currently pregnant, preparing to 
become pregnant during the study, or breastfeeding. 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
• 
Those that have undergone a change in hormone-based therapies such as, but not limited to, 
oral contraceptive pills or progesterone pills within the last 2 months. Progesterone releasing 
IUDs are considered hormone-based therapy. Spironolactone is not considered a hormone-
based therapy.   
• 
Subjects who are unwilling or unable to comply with the requirements of the protocol.  
• 
Subjects who have a history of or a current psychological illness or condition that would 
interfere with their ability to understand and follow the requirements of the study in the opinion 
of the principal investigator.  
• 
Subjects with or who have recently experienced traumatic injury, infections, or major surgery at 
the discretion of the qualified investigator. 
• 
Subjects who are likely to start taking drugs/medication on a continuous basis or that will 
undergo surgery during the trial.  
• 
Subjects engaged in donation or who were recipients of blood products within 90 days before 
the start of the study.  
• 
Subjects with alcohol use of more than 2 alcoholic beverages per day within the past month.  
• 
Subjects participated in a clinical trial with a medicinal, supplemental, nutraceutical or drug 
within the past two months prior to the first dose in the current study.  
• 
. If participants take fish oil supplements, they will be asked to stop taking these supplements 
21 days before the start of trial and during the trial.  
• 
Subjects using anti-inflammatory drugs on a chronic basis (e.g. aspirin, ibuprofen, diclofenac, 
celecoxib, etc).  
• 
Subjects using supplements or substances present in formulation (malate, fisetin, 
glucosamine, alpha ketoglutaric acid, glycine, theanine, Rhodiola rosea, hyaluronic acid, ginger 
extract, pterostilbene, lithium). Participants will be asked to stop taking these supplements 21 
days before the start of trial and during the trial.  
• 
Subjects using supplements or substances similar as those used in the formulation (e.g. 
resveratrol (similar to pterostilbene) or quercetin (similar to fisetin). Participants will be asked to 
stop taking these supplements 21 days before the start of trial and during the trial.  
• 
Subjects taking NAD boosters, like NMN, nicotinamide riboside (NR), high-dose niacin 
(nicotinic acid), high-dose vitamin B3, high-dose nicotinamide (niacinamide) within 21 days 
before the start of the trial. Participants will be asked to stop taking these supplements 21 days 
before the start of trial and during the trial.  . 
• 
Subjects with clinically significant abnormal laboratory results at screening. 
• 
Known allergy/intolerance to any of the components in the supplement product.  
Consent can be withdrawn at any time. In this case, all personal and research data will be deleted. After 
data have been anonymised, only personal data will be deleted and it will not be possible to delete 
research data after this point.  
9. Experimental design, data collection and methods (including data analysis) 
In order to assess the vascular effects of the supplement mix, we will perform a 2 armed parallel group, 
randomised, controlled, double blind clinical trial. Healthy subjects will be recruited and after reviewing 
a participant information sheet and signing the informed consent document randomised to either the 
supplement or placebo and consume it daily over 6 months (3-6 months ±1month to ensure completion 
of study and allow flexibility for participants). The randomisation will be based on a sequential list with 
equal numbers of treatments A and B, with one being placebo and one verum. Recruited participants 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
will also be numbered sequentially and receive the respective treatment (A or B). The containers of 
treatments will be labelled with either A or B. The identity of what was A and what was B will be stored 
in a sealed envelope in a locked cabinet in Prof. Heiss’s office. The measurements will take place in the 
morning, after an overnight fast and in the fasting stage, before and at 2 hours after the first dose and 
once in the morning fasted at 6 months. After measurements are completed, participants will be offered 
a breakfast snack and drink. Venous blood samples (<50 ml) will only be taken once on each study day.  
The primary endpoint will be a change endothelial function as assessed by flow-mediated dilation 
(FMD) with ultrasound on the brachial artery.  
Secondary endpoints will be change in blood pressure and arterial stiffness as determined by an upper 
arm cuff and optical sensors and ECG. Additional secondary endpoints are carotid artery stiffness, 
intima-media thickness as assessed by carotid artery ultrasound and microvascular structure and 
function with optical coherence tomography angiography (OCTA) as assessed in parallel with FMD 
measurements on the arm. As additional secondary endpoints, we will assess blood biomarkers of 
cardiovascular health including blood lipids (fasting total cholesterol, fasting LDL, fasting HDL, ApoB, 
fasting triglycerides, and fasting insulin) and glucose control (fasting glucose and HbA1c). A change in 
cardiovascular risk score will be another secondary endpoint. We will used the Heart SCORE2 estimate 
10-year risk of a myocardial infarction, stroke or cardiovascular death, based on age, sex, smoking 
habits, systolic blood pressure and non-HDL cholesterol (https://www.escardio.org/Education/Practice-
Tools/CVD-prevention-toolbox/HeartScore). A pre-specified subgroup analysis will be performed in 
people with intermediate-high (<1% estimated 10-year cardiovascular risk) and high (>5% estimated 10 
year cardiovascular risk) baseline CV risk. We expect more pronounced effects of the supplement in 
people with higher risk i.e. higher blood pressure or cholesterol at baseline. Exploratory tertiary 
endpoints will include blood biomarkers of inflammation (high sensitivity C-reactive protein, TNF-alpha 
receptor II, Interleukin 6), platelet reactivity, DNA damage (measured via gH2AX in blood and 8-
hydroxydeoxyguanosine in urine), ageing (growth differentiation factor 15, adiponectin, leptin), liver 
(transaminases) and kidney function (creatinine, cystatin C). We also want to explore novel biomarkers 
of aging, including epigenetic and transcriptomic aging clocks (on DNA samples), RNA and protein 
expression. In this regard, we will ask participants to allow us to store surplus blood, serum, plasma, 
RNA and DNA samples for future studies. To explore potential effects on dental health, we will take a 
digital photo of the teeth. Funding for the analyses needs to be obtained separately in the future. Sample 
processing and storage will comply with HTA regulations (see attached STAMINA_Human Tissue 
Governance Application Form V1.0.docx). Most blood and urine biomarkers will be analysed by Affinity 
Biomarker Lab. Governance including MTA is currently being prepared. The measurements (except for 
venous blood draws) will be entirely non-invasive, pain-free and require a single session of 
approximately 3 hours on the first day and 1 hour at 6 (3-6) months. Additional exploratory endpoints 
will be physical activity based on step/distance covered software on participants cell phone if they have 
this, dietary habits as assessed by food frequency questionnaire and 24 food recall and quality of life 
based on standard SQL questionnaire, and the completion at the start and end of the study of 8 
questionnaires that assess anxiety, depressive feelings, happiness and well-being, given various 
substances in the formulation could also impact brain health and could contribute to improved cognitive 
and mental functioning, such as magnesium, glycine and Rhodiola rosea. All questionnaires are in the 
appendix. Pre-specified subgroup analyses will be performed in people with healthier diet, estimated 
high polyphenol intake at baseline and very high physical activity. These people may exhibit a smaller 
effect of the supplement. 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
 
Composition of supplement and placebo 
The supplement mix is supplied as a powder to be mixed in water or other beverages like tea or coffee. 
The powder contains 12 ingredients, and it is commercially available (see also https://novoslabs.com/). 
All components are also part of a natural diet or are found naturally in the body. The supplement and 
matched placebo will be provided by AgeLess Sciences.  
The composition is detailed in the table below. The placebo looks and tastes similar but does not contain 
the 12 supplements ingredients, only excipients and flavours.  
Participants will receive a jar containing powder (supply of 6 months) with a spoon. Participants add one 
spoon of powder to 1 glass of water (240 ml), mix it and immediately drink it in the morning with 
breakfast. The participants will receive a paper diary to record at what time the supplement was 
consumed and if alone or with breakfast. If a dose is missed in the morning, they should just take it once 
they remember during the same day. If on a day the dose is missed entirely, we ask the participants to 
document this and carry on with the same amount on the next days but not take more than usual.   
 
Table 1: Composition of one serving size (15 g) of supplement mix and placebo. 
 
VERUM 
PLACEBO 
 
Supplement 
mix 
 
Vitamin C (ascorbic acid) 
100 mg 
0 
Magnesium (as magnesium malate) 
304 mg 
0 
Calcium (as calcium alpha-ketoglutarate) 
231 mg 
0 
Glycine 
2,000 mg 
0 
Malate (as magnesium malate) 
1,700 mg 
0 
Calcium alpha-ketoglutarate 
1,100 mg 
0 
GreenGrown® glucosamine sulfate (vegan) 
1,000 mg 
0 
Rhodiola rosea root extract (3% rosavins and 1% salidrosides) 
300 mg 
0 
L-Theanine 
150 mg 
0 
Hyaluronic acid 
100 mg 
0 
Fisetin 
100 mg 
0 
Organic ginger root extract (2% gingerols) 
100 mg 
0 
Pterostilbene 
50 mg 
0 
Lithium aspartate (providing 1 mg lithium) 
20 mg 
0 
Other ingredients: Erythritol, malic acid, natural flavours, 
sodium bicarbonate, silicon dioxide, calcium silicate, stevia 
X 
X 
 
 
 
 
“Overdosing” and adverse events 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
The recommended dose of the study supplement is 15 g which contains for example 100 mg Vit. C 
(approx. recommended daily intake 100 mg) and Lithium 1 mg (average daily intake). It is unlikely that 
participants will consume above the recommended dose as the supplement is insoluble in water if 
added in amounts above 15 g. However, to mitigate the unlikely occurrence of overconsumption, the 
following mitigation plan will be followed:  
- A diary will be completed to document the ingested amount every day and advise on not to take 
more than recommended. 
- Training on preparing the solution will be conducted by the investigator staff on the first study day 
Participants will be advised to call the investigator if they have consumed above the recommended 
dose or if any adverse event independent of dose occurs. The investigator will carefully assess any 
adverse events and advice accordingly and perform unblinding if required.  
 
Sample size justification 
The sample size is based on the primary endpoint FMD and the key secondary endpoints pulse wave 
velocity (PWV) and microvascular diameter. 
Our previous experiments have shown average FMD values of 6% with standard deviation of 1%. While 
the supplement has not been tested in terms of effects on FMD, we expect a change in 1%. To detect 
this, we will need to study 17 verum subjects and 17 placebo subjects to be able to reject the null 
hypothesis that the population means of the verum and placebo groups are equal with probability 
(power) 0.8. The Type I error probability associated with this test of this null hypothesis is 0.05. 
Our previous experiments have shown average PWV values of 9.3 m/s with the standard deviation of 
1.3 m/s. While the supplement has not been tested in terms of effects on PWV, we expect a change in 
PWV of 1 m/s. To detect this, we will need to study 28 verum subjects and 28 placebo subjects to be 
able to reject the null hypothesis that the population means of the placebo and control groups are equal 
with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis 
is 0.05. 
Our previous experiments have shown on average of microvascular diameter using OCTA of 48 µm 
with standard deviation is 6.4 µm. While the supplement has not been tested in terms of effects on 
microvascular diameter, we expect a change in diameter of 5 µm. To detect this, we will need to study 
27 verum subjects and 27 placebo subjects to be able to reject the null hypothesis that the population 
means of the verum and placebo groups are equal with probability (power) 0.8. The Type I error 
probability associated with this test of this null hypothesis is 0.05. 
Taken together, and taking 5-10% attrition into account we plan to recruit a total sample size of 60 (30 
verum and 30 placebo).  
 
Table 2: Schedule of events 
Procedures 
Visits (insert visit numbers as appropriate) 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
Screening 
(Visit 1) 
Baseline 
(Visit 1) 
Month 2 
(Phone) 
Month 4 
(Phone) 
6 (3-6±1) 
Months 
(visit 2) 
Informed consent 
x 
 
 
 
 
Screening/medical history 
x 
 
 
 
 
Randomisation 
 
x 
 
 
 
Demographics 
 
x 
 
 
 
Questionnaires 
 
x 
x 
x 
x 
Vascular exam 
 
x x 
 
 
x 
Blood sampling 
 
x 
 
 
x 
Spot urine 
 
x 
 
 
x 
Treatment (supplement or 
placebo) 
 
x 
 
 
 
 
 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
10. Risk Assessment 
1. Identified Risks 
2. 
Likelihood 
3. 
Potential Impact/ 
Outcome 
4. 
Potential 
Severity of 
Outcome 
5. 
Risk Management/Mitigating Factors 
Identify 
risks/hazards 
present 
Identify how likely 
the event is i.e. 
 
Very likely/ Likely/ 
Possible/ Unlikely 
Who might be harmed and 
how? 
 
Ensure you have considered 
the research team, participants 
and anyone not directly 
involved in the research. 
Classify the severity 
of outcomes 
identified in 3. 
 
i.e. High/ Medium/ 
Low 
Evaluate the risks and 
decide on the precautions. 
Standard 
Operating 
Procedures*/ risk 
assessments 
 
Enter Ref no/ title/ 
expiry date 
Data loss 
 
Unlikely 
The investigators and 
funders. Bad reputation for 
University and research 
team. 
High 
Data will be backed up 
and paper printout of 
CRFs stored. 
 
Data breach 
 
Unlikely 
The participants and 
investigators. Bad 
reputation for University 
and research team. 
High 
Data will be store only 
with anonymous ID and 
only with “age” but not 
DOB in password 
protected file on 
university computers and 
drives. Paper copies 
stored in locked cabinet. 
 
 
 
 
 
 
 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
1. Identified Risks 
2. 
Likelihood 
3. 
Potential Impact/ 
Outcome 
4. 
Potential 
Severity of 
Outcome 
5. 
Risk Management/Mitigating Factors 
Incidental 
findings of 
medical 
condition 
 
Possible 
High blood pressure, 
cholesterol or glucose or 
other unexpected abnormal 
biochemical measurements 
could possibly be detected. 
Participant may require 
treatment. 
Low (this may 
actually be a 
benefit for the 
participant as this 
may otherwise not 
have been 
detected) 
PI will inform participant 
and offer to personally 
inform the GP to initiate 
treatment. 
 
“Overdosing” 
 
Unlikely as 
supplement is not 
soluble in water in 
higher amounts. 
Upset stomach 
Low (no toxicity for 
components 
reported even in 
10xamounts) 
- 
Diary with 
instruction 
- 
Training by 
investigators how 
to prepare drinks 
 
 
 
 
 
 
 
 
 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
11. Data Management 
A paper list linking study ID with study subjects will be kept in a locked cabinet in Professor Heiss’s 
office (14AY04). Personal data including name, age, sex and contact details will be stored to allow 
participation in future studies. In addition, personal data will be stored in accordance with HTA 
regulations. All data will only be stored on secure university servers. Research data consisting of study 
measurements and observations will be anonymised. Research data will be shared among the 
participating researchers named above to allow analyses for publication. The anonymised data and 
blood samples may also be used for future research by the participating researchers. While we do 
currently not plan to make the anonymised and non-identifiable research data publicly available, we 
will ask participants for consent to allow us to do so in the future. 
 
12. Ethical considerations 
In case any medically relevant findings are incidentally discovered, these findings will in the first 
instance presented to Prof. Heiss, who is a consultant in the NHS, to evaluate the validity and 
potential meaning. For instance, in healthy people elevated blood pressure, cholesterol or glucose 
could be discovered that would potentially require treatment. He will then personally discuss the 
findings with the participant and offer to inform the GP if the participant wishes.  
 
13. Dissemination 
We are planning on presenting the findings in seminars at the university including SHARP network, 
national and international conferences. In addition, the results will be published in scientific journals. 
14. References 
 
1 
Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. 
Cell 153, 1194-1217, doi:10.1016/j.cell.2013.05.039 (2013). 
2 
Heiss, C., Spyridopoulos, I. & Haendeler, J. Interventions to slow cardiovascular aging: Dietary 
restriction, drugs and novel molecules. Exp Gerontol 109, 108-118, 
doi:10.1016/j.exger.2017.06.015 (2017). 
3 
Sesso, H. D. et al. Effect of cocoa flavanol supplementation for prevention of cardiovascular 
disease events: The COSMOS randomized clinical trial. Am J Clin Nutr, 
doi:10.1093/ajcn/nqac055 (2022). 
4 
Bondonno, N. P. et al. Flavonoid intake is associated with lower mortality in the Danish Diet 
Cancer and Health Cohort. Nat Commun 10, 3651, doi:10.1038/s41467-019-11622-x (2019). 
5 
Bell, G. A., Kantor, E. D., Lampe, J. W., Shen, D. D. & White, E. Use of glucosamine and 
chondroitin in relation to mortality. Eur J Epidemiol 27, 593-603, doi:10.1007/s10654-012-
9714-6 (2012). 
6 
Pocobelli, G. et al. Total mortality risk in relation to use of less-common dietary supplements. 
Am J Clin Nutr 91, 1791-1800, doi:10.3945/ajcn.2009.28639 (2010). 
7 
Ma, H. et al. Association of habitual glucosamine use with risk of cardiovascular disease: 
prospective study in UK Biobank. BMJ 365, l1628, doi:10.1136/bmj.l1628 (2019). 
8 
Duan, R., Xie, H. & Liu, Z. Z. The Role of Autophagy in Osteoarthritis. Front Cell Dev Biol 8, 
608388, doi:10.3389/fcell.2020.608388 (2020). 
9 
Weimer, S. et al. D-Glucosamine supplementation extends life span of nematodes and of 
ageing mice. Nat Commun 5, 3563, doi:10.1038/ncomms4563 (2014). 
10 
Miller, R. A. et al. Glycine supplementation extends lifespan of male and female mice. Aging 
Cell 18, e12953, doi:10.1111/acel.12953 (2019). 
11 
Obata, F. & Miura, M. Enhancing S-adenosyl-methionine catabolism extends Drosophila 
lifespan. Nat Commun 6, 8332, doi:10.1038/ncomms9332 (2015). 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
12 
Ding, Y. et al. Plasma Glycine and Risk of Acute Myocardial Infarction in Patients With 
Suspected Stable Angina Pectoris. J Am Heart Assoc 5, doi:10.1161/JAHA.115.002621 
(2015). 
13 
Guasch-Ferre, M. et al. Metabolomics in Prediabetes and Diabetes: A Systematic Review and 
Meta-analysis. Diabetes Care 39, 833-846, doi:10.2337/dc15-2251 (2016). 
14 
Cruz, M. et al. Glycine treatment decreases proinflammatory cytokines and increases 
interferon-gamma in patients with type 2 diabetes. J Endocrinol Invest 31, 694-699, 
doi:10.1007/BF03346417 (2008). 
15 
Gannon, M. C., Nuttall, J. A. & Nuttall, F. Q. The metabolic response to ingested glycine. Am J 
Clin Nutr 76, 1302-1307, doi:10.1093/ajcn/76.6.1302 (2002). 
16 
de Picciotto, N. E. et al. Nicotinamide mononucleotide supplementation reverses vascular 
dysfunction and oxidative stress with aging in mice. Aging Cell 15, 522-530, 
doi:10.1111/acel.12461 (2016). 
17 
Das, A. et al. Impairment of an Endothelial NAD(+)-H2S Signaling Network Is a Reversible 
Cause of Vascular Aging. Cell 173, 74-89 e20, doi:10.1016/j.cell.2018.02.008 (2018). 
18 
Liao, B. et al. Nicotinamide mononucleotide supplementation enhances aerobic capacity in 
amateur runners: a randomized, double-blind study. J Int Soc Sports Nutr 18, 54, 
doi:10.1186/s12970-021-00442-4 (2021). 
19 
Zarse, K., Jabin, S. & Ristow, M. L-Theanine extends lifespan of adult Caenorhabditis elegans. 
Eur J Nutr 51, 765-768, doi:10.1007/s00394-012-0341-5 (2012). 
20 
Unno, K. et al. Theanine intake improves the shortened lifespan, cognitive dysfunction and 
behavioural depression that are induced by chronic psychosocial stress in mice. Free Radic 
Res 45, 966-974, doi:10.3109/10715762.2011.566869 (2011). 
21 
Zeng, L. et al. l-Theanine attenuates liver aging by inhibiting advanced glycation end products 
in d-galactose-induced rats and reversing an imbalance of oxidative stress and inflammation. 
Exp Gerontol 131, 110823, doi:10.1016/j.exger.2019.110823 (2020). 
22 
Petrovic, J. et al. Magnesium Supplementation Diminishes Peripheral Blood Lymphocyte DNA 
Oxidative Damage in Athletes and Sedentary Young Man. Oxid Med Cell Longev 2016, 
2019643, doi:10.1155/2016/2019643 (2016). 
23 
Fuentes, J. C., Salmon, A. A. & Silver, M. A. Acute and chronic oral magnesium 
supplementation: effects on endothelial function, exercise capacity, and quality of life in 
patients with symptomatic heart failure. Congest Heart Fail 12, 9-13, doi:10.1111/j.1527-
5299.2006.04692.x (2006). 
24 
Veronese, N. et al. Effect of magnesium supplementation on glucose metabolism in people 
with or at risk of diabetes: a systematic review and meta-analysis of double-blind randomized 
controlled trials. Eur J Clin Nutr 70, 1354-1359, doi:10.1038/ejcn.2016.154 (2016). 
25 
Heiss, C. et al. Impact of cocoa flavanol intake on age-dependent vascular stiffness in healthy 
men: a randomized, controlled, double-masked trial. Age (Dordr) 37, 9794, 
doi:10.1007/s11357-015-9794-9 (2015). 
26 
Schroeter, H. et al. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on 
vascular function in humans. Proc. Natl. Acad. Sci. USA 103, 1024-1029 (2006). 
 
 
Amendment History (to be completed for subsequent versions after initial authorisation) 
Amendment 
No. 
Protocol 
version no. 
Date issued 
Author(s) of 
changes 
Details of changes made 
1 
3 
14/11/2022 
C. Heiss 
Changed 6 (±1) month to 6 (3-6) 
months 
Changed that nicotinamide component 
of supplement is supplied as capsules 
and not as part of the powder 

STAMINA Study 
Protocol – Version 5.0 23/02/2024 
 
 
 
2 
4 
17/12/2022 
C. Heiss 
Deleted nicotinamide mononucleotide 
from protocol 
3 
5 
23/02/2024 
C. Heiss 
Added reimbursement for travel and 
time