A Double-blind Placebo-controlled Exploratory Trial to Assess the Impact of Daily Lab4P Probiotic Supplementation on Cognitive Performance and Metabolic Regulation in Overweight Young Adults With Impaired Glucose Regulation
Phase: NA
Conditions: Impaired Glucose Regulation, Impaired Glucose Tolerance (Prediabetes), Prediabetes (Insulin Resistance, Impaired Glucose Tolerance), Overweight (BMI &gt; 25), Cognitive Dysfunction, Neurovascular Coupling Mechanism and Cognitive Function
Sponsor: Leeds Beckett University

 
 
 
 
 
Pro-COG Trial Protocol 
 
 
 
Long Title: A double-blind placebo-controlled exploratory trial to assess the impact of daily 
Lab4P probiotic supplementation on Cognitive Performance and Metabolic regulation in 
Overweight Young Adults with Impaired Glucose Regulation 
 
Short Title: Probiotic impact on Cognitive performance, and metabolic outcomes in overweight 
young adults with impaired glucose regulation, shortened to Pro-COG 
 
 
 
 
 
 
LREC Reference: 152291 
Clinicaltrials.gov Number: NCT07073781 
Protocol Version: 1.2 20/03/2026 
 
 
This protocol has regard for the HRA guidance 
 
 
 
 
 
 
 
 
 
 
 
 
Leeds Beckett University 
 
 

 
 
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SIGNATURE PAGE 
The undersigned confirm that the following protocol has been agreed and accepted and that the 
Chief Investigator agrees to conduct the trial in compliance with the approved protocol and will 
adhere to the principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 
2004 (SI 2004/1031), amended regulations (SI 2006/1928) and any subsequent amendments of 
the clinical trial regulations, GCP guidelines, the Sponsor’s (and any other relevant) SOPs, and 
other regulatory requirements as amended. 
 
I agree to ensure that the confidential information contained in this document will not be used for 
any other purpose other than the evaluation or conduct of the clinical investigation without the 
prior written consent of the Sponsor 
 
I also confirm that I will make the findings of the trial publically available through publication or 
other dissemination tools without any unnecessary delay and that an honest accurate and 
transparent account of the trial will be given; and that any discrepancies and serious breaches 
of GCP from the trial as planned in this protocol will be explained. 
 
For and on behalf of the Trial Sponsor: 
 
Name: Professor Lauren Owen 
 
Position: Project Supervisor and Director of 
the Behavioural Neuroscience Lab, Leeds 
Beckett University 
 
Signature:  
 
 
 
 
Date: 20/03/2026 
 
 
Chief Investigator: 
 
Name: Lewis Hepburn 
 
Signature:  
 
 
Date: 20/03/2026 
 
KEY TRIAL CONTACTS 
Insert full details of the key trial contacts including the following 
Principal Investigator 
Lewis Hepburn  
 
Address: School of Humanities and Social Sciences  
Leeds Beckett University  

 
 
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Portland Building, City Campus, Leeds LS1 9HE 
Phone: 07541333140 
Email: L.Hepburn3449@student.leedsbeckett.ac.uk 
Project Supervisor 1 
Professor Lauren Owen   
 
Address: School of Humanities and Social Sciences  
Leeds Beckett University  
Portland Building, City Campus, Leeds LS1 9HE  
Phone: 01138121256 
Email: l.j.owen@leedsbeckett.ac.uk 
Project Supervisor 2  
Dr Steve Trangmar  
 
Address: Carnegie School of Sport  
Headingley Campus, Leeds LS6 3QQ 
Phone: 01138127405 
Email: S.Trangmar@leedsbeckett.ac.uk 
Sponsor 
Leeds Beckett University (LBU) 
Funder(s) 
Organisation Name: Cultech LTD 
 
Primary Contact: Dr Daryn Michael  
Position: Senior Research Manager 
Phone: 01639 825100  
Email: darynm@cultech.co.uk 
Address: Baglan Industrial Park, Unit 34 Aberavon Rd 
 
Organisation Name: Biotechnology and Biological Sciences 
Research Council (BBSRC) – i-NutriLife Programme 
 
Email: i-NutriLife@soton.ac.uk. 
Funding Reference Number: i-NutriLife Hub PoC_R3_012 
Key Protocol Contributors 
Lewis Hepburn 
Professor Lauren Owen 
Dr Steve Trangmar  
Dr Daryn Michael 
 
i. LIST of CONTENTS 
SIGNATURE PAGE ........................................................................................................ 2 

 
 
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KEY TRIAL CONTACTS ................................................................................................... 2 
i. LIST of CONTENTS ..................................................................................................... 3 
Ii. LIST OF ABBREVIATIONS ......................................................................................... 10 
iii. TRIAL SUMMARY .................................................................................................... 12 
iv. FUNDING AND SUPPORT IN KIND ........................................................................... 14 
V. ROLE OF TRIAL SPONSOR AND FUNDER .................................................................. 15 
Vi. PROTOCOL CONTRIBUTORS .................................................................................. 15 
viii. KEY WORDS: ........................................................................................................ 16 
Ix. DIAGRAMMATIC SCHEDULE OF VISITS AND ASSESSMENTS ...................................... 17 
1. BACKGROUND ....................................................................................................... 17 
2. RATIONALE ............................................................................................................ 22 
2.1 Assessment and management of risk .................................................................. 23 
3 OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS ............................................... 23 
3.1 Primary objective ............................................................................................... 24 
3.2 Secondary objectives ......................................................................................... 24 
3.3 Primary endpoint/outcome ................................................................................. 24 
3.4 Secondary endpoints/outcomes ......................................................................... 24 
3.4.1 Cognitive Performance Endpoints ................................................................. 24 
3.4.2 Sleep Quality Endpoints ............................................................................... 24 
3.4.3 Cardiometabolic Endpoints .......................................................................... 25 
3.4.4 Peripheral and Central Vascular Endpoints .................................................... 25 
3.4.5 Inflammatory Endpoints ............................................................................... 25 
3.4.6 Anthropometric Endpoints ........................................................................... 25 
3.4.7 Body Composition Endpoints ....................................................................... 25 
3.4.8. Gut Microbiome Diversity Endpoints ............................................................ 26 
3.4.9 Secondary Safety Endpoints ......................................................................... 26 
3.6 Table of endpoints/outcomes ............................................................................. 26 
4. TRIAL DESIGN ........................................................................................................ 28 

 
 
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5 TRIAL SETTING......................................................................................................... 28 
6 PARTICIPANT ELIGIBILITY CRITERIA .......................................................................... 28 
6.1 Inclusion criteria ................................................................................................ 28 
6.2 Exclusion criteria ............................................................................................... 29 
7 TRIAL PROCEDURES ................................................................................................ 29 
7.1 Recruitment ...................................................................................................... 29 
7.1.1 Participant identification .............................................................................. 30 
7.1.2 Screening Procedures .................................................................................. 31 
7.1.3 Payment ..................................................................................................... 32 
7.2 Consent ............................................................................................................ 32 
7.2.1 Additional consent provisions for collection and use of participant data and 
biological specimens in ancillary studies ............................................................... 33 
7.3 The randomisation scheme ................................................................................ 34 
7.3.1 Method of implementing the randomisation/allocation sequence ................... 34 
7.4 Blinding............................................................................................................. 34 
7.5 Emergency Unblinding ....................................................................................... 35 
7.6 Baseline Data .................................................................................................... 36 
7.6.1 Participant Reported Data ............................................................................ 36 
7.6.2 Cognitive Performance Data ......................................................................... 36 
7.6.3 Clinical Assessment Data............................................................................. 36 
7.7 Trial assessments .............................................................................................. 36 
7.7.1 Testing Visit 2 (Week 6) ±3 days ..................................................................... 36 
7.7.2 Testing Visit 3 (Week 11) ±3 days ................................................................... 37 
7.7.3 Testing Visit 4 (End of Intervention, Week 12) ±3 days ..................................... 37 
7.8 Withdrawal criteria ............................................................................................ 37 
7.8.1 Participant-Initiated Withdrawal ................................................................... 37 
7.8.2 Investigator-Initiated Withdrawal .................................................................. 37 
7.8.3 Scope of Withdrawal .................................................................................... 38 

 
 
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7.8.4 Documentation and Follow-Up ..................................................................... 38 
7.8.5 Investigational Product Discontinuation ........................................................ 38 
7.8.6 Replacement of Withdrawn Participants ........................................................ 38 
7.8.7 Trial Termination Criteria .............................................................................. 38 
7.9 End of trial ......................................................................................................... 38 
8 MATERIALS AND MEASURES .................................................................................... 39 
8.1 Participant reported data ................................................................................... 39 
8.1.1 Health Events and Physical Experiences ........................................................ 39 
8.1.2 Sleep Quality ............................................................................................... 39 
8.2.3 Demographics and Lifestyle ......................................................................... 40 
8.1.4 Dietary Habits ............................................................................................. 40 
8.2 Cognitive Performance ....................................................................................... 40 
8.2.1 Memory Tasks ............................................................................................. 40 
8.2.2 Executive Function Tasks ............................................................................. 41 
8.2.3 Attention and psychomotor speed Tasks ....................................................... 42 
8.3 Laboratory assessments .................................................................................... 42 
8.3.1 Cardiometabolic data .................................................................................. 42 
8.3.1.1 Blood Glucose ....................................................................................... 42 
8.3.1.2 Glucose Tolerance ................................................................................. 42 
8.3.1.3 Glycated Hemoglobin (HbA1c) ................................................................ 42 
8.3.1.4 Lipid Profile ........................................................................................... 43 
8.3.1.5 Blood Pressure ...................................................................................... 43 
8.3.2 Anthropometric data .................................................................................... 43 
8.3.3 Body Composition data ................................................................................ 43 
8.3.4 Peripheral and central vascular function ....................................................... 44 
8.3.4.1 Cerebrovascular Reactivity (CVR)............................................................ 44 
8.3.4.2 Flow-Mediated Dilation (FMD) ................................................................. 44 
8.4 Clinical sample collection procedures ................................................................ 44 

 
 
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8.4.1 Venous blood collection ............................................................................... 45 
8.4.2 Stool sample collection ................................................................................ 45 
8.5 Storage and analysis of clinical samples.............................................................. 45 
8.5.1 Venous Blood Samples ................................................................................ 46 
8.5.1.1 Processing and storage .......................................................................... 46 
8.5.1.2 Analysis ................................................................................................ 46 
8.5.1.3 Documentation and Destruction ............................................................. 46 
8.5.2 Capillary Blood Samples .............................................................................. 46 
8.5.2.2 Processing and storage .......................................................................... 47 
8.5.2.3 Analysis ................................................................................................ 47 
8.5.3 Stool Samples ............................................................................................. 47 
8.5.3.2 Processing and storage .......................................................................... 47 
8.5.3.2.1 Shotgun metagenomic sequencing ................................................... 47 
8.5.3.3 Analysis ................................................................................................ 47 
8.5.3.4 Taxonomic profiling ............................................................................ 47 
8.5.3.4 Documentation and Destruction ............................................................. 48 
9. TRIAL TREATMENTS................................................................................................. 48 
9.1 Name and description of product under investigation ........................................... 48 
9.2 Storage and supply ............................................................................................ 48 
9.2.1 Storage and shipment to sites ....................................................................... 48 
9.2.2 Accountability ............................................................................................. 49 
9.2.3 Storage at trial site ....................................................................................... 49 
9.2.4 Transportation and storage at subject home .................................................. 49 
9.2.5 Return and destruction of product ................................................................ 49 
9.3 Preparation and labelling of Investigational Product ............................................. 49 
9.4 Dosage schedules ............................................................................................. 50 
9.4.1 Missed Doses .............................................................................................. 50 
9.4.2 Vomiting Post-Dose ..................................................................................... 50 

 
 
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9.4.3 Treatment Resumption After Temporary Suspension ...................................... 50 
9.5 Known reactions and interaction with other therapies .......................................... 51 
9.6 Concomitant medication ................................................................................... 51 
9.7 Trial restrictions ................................................................................................. 51 
9.8 Assessment of compliance with treatment .......................................................... 52 
10 PHARMACOVIGILANCE .......................................................................................... 52 
10.1 Definitions....................................................................................................... 52 
10.2 Operational definitions for (S)AEs ...................................................................... 53 
10.2.1 Identification of (S)AEs ............................................................................... 53 
10.2.2 Evaluation of AEs and SAEs ......................................................................... 53 
10.2.2.1 Severity ............................................................................................... 54 
10.2.2.2 Causality ............................................................................................. 54 
10.2.2.3 Expectedness ...................................................................................... 54 
10.2.3 Events to Be Reported to the Sponsor .......................................................... 54 
10.2.4 Events Not Requiring Reporting to the Sponsor ............................................. 55 
10.3 Recording and reporting of SAEs, SARs AND SUSARs .......................................... 55 
10.4 Responsibilities ............................................................................................... 56 
10.5 Notification of deaths ....................................................................................... 56 
10.6 Pregnancy reporting ......................................................................................... 57 
10.7 Reporting urgent safety measures ..................................................................... 57 
10.8 The type and duration of the follow-up of participants after adverse reactions. ..... 57 
11 STATISTICS AND DATA ANALYSIS ............................................................................ 57 
11.1 Sample size calculation.................................................................................... 57 
11.2 Planned recruitment rate .................................................................................. 57 
11.3 Statistical analysis plan .................................................................................... 58 
11.3.1 General ..................................................................................................... 58 
11.3.2 Summary of baseline data and flow of patients ............................................ 58 
11.3.3 Primary outcome analysis .......................................................................... 58 

 
 
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11.3.4 Secondary outcome analysis ...................................................................... 59 
11.3.5 Microbial Diversity analysis......................................................................... 59 
11.3.5.1 Alpha Diversity ..................................................................................... 59 
11.4 Subgroup analyses........................................................................................... 60 
11.5 Adjusted analysis ............................................................................................. 60 
11.6 Interim analysis and criteria for the premature termination of the trial .................. 60 
11.7 Participant Population ...................................................................................... 61 
11.7.1 Full analysis dataset .................................................................................. 61 
11.7.2 Per-Protocol analysis dataset ..................................................................... 61 
11.7.3 Safety analysis set ..................................................................................... 61 
11.8 Procedure(s) to account for missing or spurious data ......................................... 61 
11.9 Other statistical considerations. ....................................................................... 61 
12. DATA MANAGEMENT ............................................................................................. 61 
12.1 Data collection tools and source document identification .................................. 61 
12.1.2 Source documents ..................................................................................... 62 
12.1.2.1 Investigator’s site data ......................................................................... 62 
12.1.2.2 Patient reported outcomes ................................................................... 62 
12.1.2.3 External data and laboratory data .......................................................... 62 
12.1.2.4 Cognitive Assessment Data (CANTAB Platform) ..................................... 62 
12.1.2.5 Case Report Forms as source Documents ............................................. 62 
12.2 Data handling and record keeping ..................................................................... 63 
12.2.1 Audit trail and eSignature ........................................................................... 63 
12.2.2 Data validation .......................................................................................... 63 
12.2.3 Coding ...................................................................................................... 63 
12.2.4 Database Lock ........................................................................................... 63 
12.3 Access to Data ................................................................................................ 63 
12.4 Archiving ......................................................................................................... 63 
13. MONITORING, AUDIT & INSPECTION ...................................................................... 64 

 
 
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14. ETHICAL AND REGULATORY CONSIDERATIONS ..................................................... 64 
14.1 Research Ethics Committee (REC) review & reports ............................................ 64 
14.2 Regulatory Compliance .................................................................................... 65 
14.3 Protocol compliance ........................................................................................ 65 
14.4 Notification of Serious Breaches to GCP and/or the protocol ............................... 65 
14.5 Data protection and patient confidentiality ........................................................ 65 
14.5.1 Subject data’ rights .................................................................................... 66 
14.5.3 Data Breaches reporting ............................................................................. 66 
14.5.4 Retention of Data ....................................................................................... 67 
14.6 Financial and other competing interests ............................................................ 67 
None ...................................................................................................................... 67 
14.7 Indemnity ........................................................................................................ 67 
14.8 Amendments ................................................................................................... 67 
14.9 Post trial care .................................................................................................. 67 
14.10 Access to the final trial dataset ....................................................................... 68 
15 DISSEMINIATION POLICY ....................................................................................... 68 
15.1 Dissemination policy ........................................................................................ 68 
15.2 Authorship eligibility guidelines and any intended use of professional writers ....... 69 
16 REFERENCES ........................................................................................................ 69 
17 APPENDICIES ........................................................................................................ 82 
17.1 Appendix A: Schedule of Assessments .............................................................. 82 
17.2 Appendix B: Amendment History ....................................................................... 83 
 
 
 
Ii. LIST OF ABBREVIATIONS 
AE 
Adverse Event 
AUC 
Area Under the Curve  
BA 
Brachial Artery 
BBB 
Blood Brain Barrier 

 
 
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BG 
Blood Glucose 
BMI 
Body Mass Index 
CANTAB 
Cambridge Neuropsychological Test Automated Battery 
CFU 
Colony Forming Units 
CPM 
Clinical Project Manager 
CRA 
Clinical Research Associate (synonym: trial monitor) 
CRE 
Clinical Radiation Expert  
CRF 
Case Report Form 
CSS 
Carnegie School of Sport 
CV 
Coefficient of Variation 
CVR 
Cerebrovascular reactivity 
DMC 
Data Monitoring Committee  
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
EDQF 
Electronic Data Query Forms 
EDTA 
Ethylenediaminetetraacetic acid 
ELISA 
Enzyme-Linked Immunosorbent Assay 
eTMF 
Electronic Trial Master File 
FMD 
Flow-Mediated Dilation 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation 
GI 
Gastrointestinal 
GLP-1 
Glucagon-like peptide-1  
HbA1c 
Glycated Haemoglobin A1c 
HDL-C 
High Density Lipoprotein Cholersterol 
HRA 
Health Research Authority  
ICH 
International Conference on Harmonization  
ICMJE 
International Committee of Medical Journal Editors 
iDXA 
Dual-energy X-ray Absorptiometry 
IEC 
Independent Ethics Committee 
IED 
Intra-Extra Dimensional Set Shift Task 
IGM 
Impaired Glucose Metabolism 
IGT 
Impaired Glucose Tolerance 
IR 
Insulin Resistance 
IRMER 
Ionising Radiation Medical Exposure Regulations 
IL-6 
interleukin-6 
IP 
Investigational Product 
IRB 
Institutional Review Board 

 
 
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ITT 
Intent-To-Treat 
LBU 
Leeds Beckett University  
LDL-C 
Low Density Lipoprotein Cholersterol 
LSAF 
Life Science Analytics Framework 
MCA 
Middle Cerebral Artery 
MD 
Medical Doctor 
MOT 
Motor Screening Task 
MPE 
Medical Physics Expert 
OGTT 
Oral Glucose Tolerance Test 
PM 
Project Manager 
PAL 
Paired Associates Learning Task 
PI 
Principal Investigator  
PIS 
Participant Information Sheet 
PP 
Per-protocol 
PSQI 
Pittsburgh Sleep Quality Index 
REC 
Research Ethics Committee 
RNV 
Recommended Nutrient Value 
RTI 
Five Choice Reaction Time task 
RTSM 
Randomisation Trial Supply Management 
SAE 
Serious Adverse Event 
SOP  
Standard Operating Procedures 
SST 
Stop Signal Task 
SUSAR 
Serious Unexpected Suspected Adverse Reaction 
SWM 
Spatial Working Memory Task 
TFM 
Total Fat Mass 
TLM 
Total Lean Mass 
TMF 
Trial Master File 
TNF-α 
Tumor necrosis factor-alpha  
T2D 
Type 2 Diabetes 
UK 
United Kingdom 
 
iii. TRIAL SUMMARY 
TRIAL TITLE  
A double-blind placebo-controlled exploratory trial to assess the impact of 
Lab4P probiotic supplementation on Cognitive Performance and Metabolic 
regulation in Overweight Young Adults with Impaired Glucose Regulation 
TRIAL N°  
LBU-Lab4P-ProCogTrial-2025 

 
 
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TRIAL 
OBJECTIVES AND 
ENDPOINTS 
Primary objective 
 
Evaluate changes in verbal recall memory task performance from baseline to 3 
months in individuals with impaired glucose tolerance receiving Lab4P probiotic vs. 
placebo. 
Co-primary endpoints 
− 
Change in verbal episodic memory task accuracy from baseline to 3 
months. 
Secondary objectives 
− 
To evaluate the effect of Lab4P supplementation on performance in 
cognitive tasks assessing memory, executive function and psychomotor 
ability. 
− 
To evaluate the effect of Lab4P supplementation on subjective sleep 
quality. 
− 
To evaluate the impact of Lab4P on cardiometabolic health, inflammatory 
cytokine levels, body composition, and gut microbiome composition. 
Secondary endpoints  
Change in the following measures from baseline to 3 months: 
− 
Performance score on 5 cognitive tasks. 
− Cardiometabolic markers. 
− Peripheral and central vascular function. 
− 
Inflammatory cytokines. 
− 
Markers of platelet activity.  
− 
Anthropometric measures. 
− 
DXA scan-assessed body composition. 
− 
Gut microbiota community composition, dispersion and abundance of 
individual microbial taxa. 
− 
Sleep quality scores. 
Secondary Safety objectives  
To assess the tolerability and safety profile of Lab4P over 3 months. 
Secondary Safety endpoints  
− 
Adherence to investigational product. 
− 
Frequency and severity of gastrointestinal side effects.   
TRIAL DESIGN 
Double-blind, placebo-controlled, randomized parallel-group design. 
Participants will be randomly assigned to receive either the investigational 
treatment or a placebo.   
TRIAL 
POPULATION 
Healthy adults between 18-40 with Impaired Glucose Tolerance, defined by a 
glucose area under the curve (AUC), of 290 mg h/dl during an oral glucose 
tolerance test (OGTT; Sakaguchi et al., 2015). 
INCLUSION 
CRITERIA 
Must provide informed consent, have a BMI classified as overweight (25.0-
29.9kg/m2), be in good general health, report regular sleep patterns, and be able 
to complete study procedures, including fasting and OGTT. 
EXCLUSION 
CRITERIA 
Exclusion applies to those with diabetes, diagnosed sleep disorders, fasting 
glucose >6.9 mmol/L, bariatric surgery, recent major surgery or illness, 
pregnancy, or recent night shift work, taking medications that may interfere with 
probiotic activity or glucose metabolism at time of screening. 
INVESTIGATIONA
Test product:  

 
 
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L PRODUCT 
Lab4P probiotic consortium, contains a blend of the following probiotic strains; 
Lactobacillus acidophilus CUL60 (NCIMB 30157), Lactobacillus acidophilus 
CUL21 (NCIMB 30156), Lactobacillus plantarum CUL66 (NCIMB 30280), 
Bifidobacterium bifidum CUL20 (NCIMB 30153), and Bifidobacterium animalis 
subsp. lactis CUL34 (NCIMB 30172). The consortium will be delivered on a 
microcrystalline cellulose base at a total concentration of 5 × 10¹⁰ colony-forming 
units (CFU) per capsule. The intervention also contains Vitamin C, Vitamin D, and 
Zinc.  
Control product:  
Placebo capsules composed of microcrystalline cellulose, identical in appearance 
to the test product.  
MODE OF 
ADMINISTRATION 
Participants will take one capsule orally per day at any time of day. Capsules 
should be swallowed whole and not opened or chewed. Participants will be 
encouraged to take the capsule at the same time each day to support 
consistency. 
TREATMENT 
DURATION 
Participants will receive either Lab4P or placebo for a total of 12 weeks (84 
days). There is no run-in or washout period. The treatment period concludes 
with a follow-up visit at the end of week 12. 
ADMINISTRATION 
SCHEME 
Capsules will be dispensed in sealed, pre-labelled containers at the baseline visit. 
Unused capsules will be returned at the week 12 follow-up visit to assess 
compliance via capsule count. Participants will be instructed not to double up on 
missed doses. 
STATISTICAL 
METHODS 
Sample size: 
Based on prior probiotic studies reporting effect sizes of 0.60–0.67 for change in 
verbal memory score, a conservative average effect size of 0.63 was used. Power 
analysis (G*Power v3.1.9.7) with one-tailed α = 0.05 and 80% power yielded a 
required sample size of 64 participants (32 per group). To allow for an estimated 
10% attrition rate, up to 70 participants will be enrolled.  
Endpoint Analysis: 
The primary analysis will evaluate the effect of Lab4P probiotic supplementation 
versus placebo on verbal memory performance, measured by accuracy on the 
VRM task, from baseline to Week 12. A linear mixed-effects model will assess 
change over time, with fixed effects for time, treatment group, and their 
interaction, and a random intercept for participants.  
Secondary analyses, using data collected at three timepoints (baseline, Week 6, 
and Week 12), will be conducted using the same approach. For secondary 
outcomes measured only at baseline and 12 weeks, between-group differences 
in change will be assessed at both 6- and 12-week timepoints using delta 
scores, adjusting for baseline values. These analyses are exploratory and 
hypothesis-generating; no formal statistical adjustments will be made for 
multiple testing.    
Analyses will follow an intention-to-treat approach, with sensitivity analyses for 
protocol adherence and missing data. No adjustment for multiple comparisons 
is required as there is a single primary outcome. 
 
iv. FUNDING AND SUPPORT IN KIND 

 
 
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FUNDER 
FINANCIAL AND NON FINANCIALSUPPORT 
GIVEN 
Cultech Limited  
Unit 2 Christchurch Road, Baglan 
Industrial Estate, Port Talbot, W Glam, 
SA12 7BZ 
Cash: £ 9,097.32 
Staff time: £ 5,000 
Materials provided: £ 10,000 
BBSRC i-NutriLife Hub 
Biotechnology and Biological Sciences 
Research Council (BBSRC), part of 
UKRI 
i-NutriLife Hub PoC_R3_012 
Cash: £24,097.32 
 
V. ROLE OF TRIAL SPONSOR AND FUNDER 
Sponsor: 
 Leeds Beckett University will act as the Sponsor for this study. The Sponsor holds overall 
responsibility for the initiation, management, and financing (or arrangement of financing) of the 
research. This includes ensuring that appropriate systems are in place for the management, 
monitoring, and reporting of the study. While the Sponsor may delegate specific duties to 
individuals or organisations as appropriate, it retains overall accountability.  
 
Funder: 
Cultech LTD is providing financial support for the study and will contribute expert input 
specifically related to the handling of the probiotic intervention, as well as the design, logistics, 
and analysis of gut microbiome outcomes. They will have no involvement in the overall study 
design, data collection (outside of microbiome analysis), outcome measurement, interpretation 
of findings, or dissemination. The academic research team will lead all other aspects of the 
study independently and will comply fully with the funder’s Terms and Conditions. 
 
The BBSRC i-NutriLife grant supports academic research infrastructure and activities. 
Funding includes directly incurred costs (staff time, travel/subsistence, consumables, other non-
equipment costs), as well as directly allocated contributions for project team members. The 
BBSRC has no involvement in the study’s design, conduct, analysis, or dissemination. 
 
Vi. PROTOCOL CONTRIBUTORS  
This protocol was developed as part of a doctoral research programme and reflects input from a 
multidisciplinary team with specialist expertise in clinical trial design, cognitive neuroscience, 
cardiovascular physiology, and microbiome science. The study design has been shaped by 
academic supervisors with internationally recognised expertise in human intervention research, 
particularly in the neurocognitive and physiological effects of dietary and bioactive compounds. 
Contributions from an industry collaborator have supported the development and 
implementation of the probiotic intervention, microbiome analyses and randomisation process.  

 
 
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Lewis Hepburn - PhD student, Principal Investigator 
Lead author of the protocol and leader of the overall design of this trial. Responsible for the 
development and coordination of the study, drafting of all protocol sections, and integration of 
feedback from collaborators.  
Professor Lauren Owen - Primary Academic Supervisor 
Provided strategic oversight the entire trial design and development process. Professor Owen’s 
contributions included critical expert input on the formulation of research questions, study 
methodology, participant recruitment strategy, and outcome selection. Professor Owen also 
ensured that the protocol meets both academic standards and ethical requirements, and her 
supervision has ensured the scientific robustness and feasibility of the project. 
Dr Steve Trangmar: - Secondary Academic Supervisor 
Reviewed the protocol and contributed to the conceptual and practical aspects of trial planning 
alongside the primary academic supervisor. Dr Trangmar provided expert input on the vascular 
and haemodynamic components of the trial, including the selection of relevant outcome 
measures and data acquisition protocols.  
Dr Daryn Michael: - Industry Collaborator Primary Contact, Senior Research Manager 
Contributed specialist input on the interventional product and placebo, including formulation, 
supply logistics, and safety profile. Also advised on the design and analytical approach to gut 
microbiome sampling and data analysis. As the industry partner will be responsible for 
managing randomisation, Dr Michael provided guidance on the randomisation strategy to 
ensure the process is operationally feasible, blinded as appropriate and compliant with trial 
integrity standards. 
viii. KEY WORDS: 
Probiotic supplementation 
Psychobiotic  
Cognitive performance  
Memory improvement  
Metabolic function  
Impaired glucose tolerance 
 
 

 
 
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Ix. DIAGRAMMATIC SCHEDULE OF VISITS AND ASSESSMENTS 
 
 
1. BACKGROUND 
An estimated 2.11 billion adults are affected by overweight and obesity (Ng et al., 2025). The 
metabolic health consequences linked to excess body weight are well established. Overweight 
and obesity are consistently associated with a higher prevalence of impaired glucose 
metabolism (Power & Thomas, 2011). Disruptions in glucose regulation are both characteristic 
of and contributory to the pathogenesis of metabolic disorders, including type 2 diabetes (T2D), 
metabolic syndrome. Clinical perturbations in glucose homeostasis are strongly associated with 
sleep disturbances, cognitive impairments, and an increased risk of neurodegenerative 
diseases (Marseglia et al., 2019; Procaccini et al., 2016). Increasing evidence suggests that 
even slightly impaired glucose metabolism (IGM) may contribute to cognitive decline (Kirvalidze 
et al., 2022). Under normal physiological conditions, glucose serves as the primary energy 
substrate for nearly all brain cells, particularly neurons, which have a high demand for 
continuous energy supply, but limited capacity for fuel storage (Faria-Pereira & Morais, 2022; 
Ritter, 2017). The regulation of brain glucose metabolism is tightly controlled by the 
neurovascular unit (NVU), a coordinated system comprising neurons, astrocytes, endothelial 
cells, pericytes, and microglia (Takahashi, 2020). Neuronal activation prompts the NVU to 
increase cerebral blood flow (CBF) through coordinated vasodilation, ensuring adequate energy 
supply (Kaplan et al., 2020; Takahashi, 2022). Thus, the NVU solves the brains paradoxical 
need for sustained energy supply despite low storage capacity by regulating the precise delivery 
of glucose, and other metabolic substrates.  
  
Cerebral glucose metabolism changes throughout the lifespan, alongside brain development. In 
healthy individuals, cognitive ability undergoes rapid development throughout childhood, 
reaches peak efficiency in early adulthood, and declines gradually across most domains from 
mid to late adulthood (Nichols et al., 2021). Central and peripheral glucose metabolism follows a 
similar trajectory; with cerebral metabolic rate and overall glucose requirements peaking in early 

 
 
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childhood to support development, gradually declining throughout childhood and adolescence, 
before stabilizing in late adolescence (Iozzo & Guzzardi, 2019). This stability persists until 
middle age, when metabolism begins to deteriorate (Assuncao et al., 2018; Tan et al., 2011).   
Given the brain's reliance on glucose for energy, it is unsurprising that midlife represents a 
critical period in brain aging, demonstrating a slow decline in cognition and neurostructural 
alterations (Aalst et al., 2021; Carmichael et al., 2019). Older adults with prediabetes have been 
shown to exhibit significantly reduced memory retention and executive functioning compared to 
normoglycemic individuals (Hoyos et al., 2022). IGM in older adults is associated with deficits in 
global cognition, executive function, processing speed, and working memory (Lamport et al., 
2009; Mortby et al., 2013). In addition to reductions in cognitive performance, glycaemic 
dysregulation is also associated with alterations in both structural and functional brain integrity. 
For example, neuroimaging studies in adults above the age of 50 have associated IGM with 
reductions in hippocampal and temporal lobe volume, increased neurovascular burden, and 
decreased total brain volume (Jang et al., 2024; Oh et al., 2021). Some research has suggested 
that the hippocampus, associated with spatial awareness, and both long- and short-term 
memory consolidation is particularly susceptible to glycaemic dysregulation in older adults 
(Sevim et al., 2024; Soleymani et al., 2024; Zhong et al., 2020). Thus, the brain appears to 
exhibit region-specific structural and functional vulnerability in response to declining metabolic 
homeostasis, changes that become increasingly pronounced by midlife.   
  
While most evidenced in mid to older adulthood, neurocognitive deficits are also observed 
alongside IGM in adults below the age of 40 (Lamport et al., 2009; Soleymani et al., 2024). The 
prevalence of metabolic dysregulation in this age group has risen markedly in recent decades 
and is projected to continue its upward trajectory (Chong et al., 2023; Rooney et al., 2023). 
Indeed, epidemiological data indicate that markers of IGM may be detectable in as high as 24% 
of individuals aged 18 to 37 (Andes et al., 2019; Madeira et al., 2013; Magliano & Boyko, 2021). 
Research is increasingly demonstrating that dysregulation impacts cognition across multiple 
domains. Elevated fasting BGL, and HbA1c in young, overweight and obese individuals have 
been associated with lower working memory, reading ability, and executive function (Hawkins et 
al., 2016; Hawkins et al., 2021). Longitudinal evidence reinforces this pattern, with higher HbA1c 
in non-diabetic younger men associated with poorer processing speed and both immediate and 
delayed recall ability (Anstey et al., 2015). Similarly, higher-normal fasting BGL appears to 
predict impaired inhibitory control, spatial perception, and selective attention (Gluck et al., 2013; 
Hawkins et al., 2016; Razzak et al., 2018). In addition to static measures of IGM, dynamic 
indices of glycaemic regulation, such as Oral Glucose Tolerance Testing (OGTT) are also 
associated with cognition in young adults. For example, IGM assessed via postprandial glucose 
handling has been associated with poorer verbal memory performance, and impaired behavioral 
flexibility, a subdomain of executive function in young, nondiabetic adults (Messier et al., 2011; 
Riby et al., 2017). Alongside direct measures of glucose, subclinical levels of IR in young adults 
are associated with lower visuomotor ability and reductions in both working and episodic 
memory performance (Abbasi et al., 2023; Larsen et al., 2023). Amid the ongoing global obesity 
epidemic, early manifestations of metabolic dysregulation, once primarily observed in midlife, 
are increasingly prevalent in early adulthood. This indicates a possible downward trend in the 
age at which cognitive vulnerability emerges, raising concern that cognitive decline may now 
begin earlier in the lifespan than historically observed.     
  
Few neuroimaging studies have focused on the consequences of IGM in early adulthood. 

 
 
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However, the limited research available suggests that the brain regions affected in this 
demographic resemble those observed in diabetes, though typically to a lesser degree. For 
instance, studies including young to midlife adults have reported age-independent associations 
between IGM and moderate structural and functional alterations in both grey and white matter, 
particularly in brain regions involved in visual processing, memory, and semantic integration 
(Grundmann et al., 2020; Shan et al., 2019; Weinstein et al., 2015). In studies involving 
exclusively young adults aged 22-35, Diffusion Tensor Imaging has revealed an association 
between elevated, within-normal HbA1c levels, and decreased markers of white matter integrity 
in areas involved in executive function, attention, motor coordination, and emotional regulation 
(Repple et al., 2019; Ribeiro et al., 2023). Indeed, Repple and colleagues (2019) found these 
decrements to mediate a relationship between HbA1c and performance in global cognition, 
working memory, and fluid intelligence tasks. These microstructural alterations in white matter 
may underlie functional connectivity weaknesses observed in similar demographics. In 
prediabetic adults aged 22-35, reduced functional connectivity in white-matter associated 
networks supporting self-control and attentional regulation have been observed (Sadler et al., 
2019; Santillo et al., 2024; Shearrer, 2023). White matter is particularly susceptible to 
cerebrovascular damage, owing to its cellular metabolic demand and relatively limited vascular 
supply (Levit et al., 2020). As functional connectivity depends on the structural integrity of white 
matter tracts, and is independently sensitive to NVU function, disruptions in both structure and 
function are likely manifestations of early NVU impairment (Guilbert et al., 2022; Nozais et al., 
2023). Collectively, these findings support the hypothesis that the cognitive impairments 
observed in young adults with IGM are, at least in part, attributable to NVU dysfunction.   
  
One proposed pathway by which IGM disrupts NVU function involves endothelial dysfunction at 
the blood-brain barrier (BBB; Lamport et al., 2009). Exaggerated glucose excursions, even 
within the subclinical IGM range, promotes excessive glucose uptake into endothelial cells, 
activating biochemical pathways that increase the generation of reactive oxygen species (ROS) 
and promote the formation of advanced glycation end products (AGEs; Di Pino et al., 2019; 
Korkmaz et al., 2013). Both ROS and AGEs contribute to oxidative stress and endothelial 
damage through mitochondrial dysfunction, redox imbalance and pro-apoptic signaling (An et 
al., 2023; Clyne, 2021). Further, this oxidative stress elicits a pro-inflammatory cellular response 
that contributes to endothelial damage, and independently disrupts neurophysiological 
processes (Collier et al., 2008; De Carvalho Vidigal et al., 2013; Theofilis et al., 2021). In 
addition to contributing to hyperglycaemia, IR further impairs BBB endothelial integrity and 
selective permeability (Cui et al., 2022; Kullmann et al., 2016). At the BBB, endothelial 
dysfunction compromises vasoactive signaling and barrier homeostasis, disrupting the NVU’s 
capacity to regulate CBF in response to neuronal activity (Feng & Gao, 2024; Toth et al., 2017). 
It has also been suggested that both IR and chronic hyperglycaemia may impair the expression 
and function of glucose transporter proteins at the BBB, further disrupting the brain's energy 
supply (Blázquez et al., 2014; Leão et al., 2020). Together, these processes may compromise 
the ability of the NVU to maintain cerebral homeostasis, leading to subtle but functionally 
meaningful disruptions in brain perfusion and energy metabolism, both of which are essential for 
supporting cognition.   
  
While metabolic dysfunction has most consistently been linked to impaired cerebrovascular 
function in T2D cohorts, importantly, similar impairments have been observed in individuals at 
earlier stages of dysregulation (Hardigan et al., 2016; Vidyashree et al., 2022). For instance, 

 
 
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reduced internal carotid artery reactivity has been reported in mid-life adults with IGT compared 
to normoglycemic controls (Selvarajah et al., 2016). Peripheral endothelial dysfunction, a proxy 
measure for central vascular health, has been reported in individuals with IGT during both early 
and midlife stages (Salmon et al., 2024; Smirnova et al., 2013; Su et al., 2008). Indeed, NVU 
dysfunction has been observed directly in young adults with IGM. Under typical conditions, CBF 
and regional cerebral glucose metabolism (CMRGlc) are tightly coupled, whereby a reduction in 
one prompts an increase in the other to maintain cerebral metabolic homeostasis (Paulson et 
al., 2010). However, Deery et al. (2024b) reported altered coupling between CBF and CMRGlc 
in young, insulin-resistant adults without overt diabetes. In a similar population, lower rates of 
regional cerebral glucose metabolism were observed in prefrontal, parietal and temporal 
regions, and was associated with lower working memory, slower reaction time and psychomotor 
speed (Deery et al., 2024a). These findings suggest that early cerebrovascular alterations in 
IGM may disrupt the NVU’s capacity to regulate and utilise glucose efficiently, with IR potentially 
contributing to these impairments. Thus, current evidence suggests that metabolically mediated 
brain microvascular dysfunction may present in young and middle-aged adults prior to the onset 
of overt metabolic pathology.  
  
The observation of a detectable decoupling between CBF and brain glucose metabolism in 
young adults with poor peripheral glucose regulation aligns with evidence that insulin 
administration significantly increases brain glucose metabolism in individuals with IGT, but not in 
healthy controls (Hirvonen et al., 2011). This indicates that cerebral insulin action is not 
saturated at fasting levels in those with IGT, potentially limiting glucose availability and insulin-
mediated neuromodulatory processes, thereby contributing to cognitive impairment (Malin et al., 
2022). Further evidence of altered cerebral glucose autoregulation in young adults with IGM 
comes from glucose administration studies, wherein cognitive performance is enhanced to a 
greater degree than normoglycaemic peers (Meikle et al., 2004; Owen et al., 2013). In these 
individuals, acute increases in glucose availability following administration likely elevate 
extracellular glucose concentrations in the brain, temporarily restoring cerebral energy supply, 
compensating for deficits in glucose transport and improving cognitive function (Duarte, 2015; 
Gold, 2005). Memory is the cognitive domain most consistently enhanced by glucose 
administration in young adults, possibly due to its reliance on medial temporal lobe regions that 
are especially sensitive to changes in glucose availability (Smith et al., 2011). This sensitivity is 
attributed to the high density of both insulin receptors and glucose transporters in these areas 
(Koepsell, 2020; Pomytkin et al., 2018). Overall, this evidence suggests that even in the early 
stages of glycaemic dysregulation, impairments in vascular-metabolic coupling are evident and 
may offer a mechanism underlying the cognitive impairment associated with IGM.  
  
Whether interventions targeting metabolic health can mitigate observed cognitive deficits is a 
question of clear importance. Current therapeutic strategies for metabolic dysfunction primarily 
involve either lifestyle modification or pharmacological treatment (Blüher et al., 2023). Growing 
interest in the gut-brain axis and its role in neurocognitive processes has led to exploration of 
probiotics as a potential alternative or adjunctive strategy to improve cognition. Systematic 
reviews and meta-analyses indicate that probiotics may offer cognitive benefits (Irwin et al., 
2020; Marx et al., 2020). Probiotic supplementation has been associated with improved mental 
flexibility in healthy older adults and to significant gains in immediate memory, delayed recall, 
and visuospatial abilities in those with mild cognitive impairment (Kim et al., 2020; Xiao et al., 
2020). Additionally, working memory and verbal learning improvements have been observed in 

 
 
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young adults under stress, following supplementation (Lew et al., 2019; Oluwagbemigun et al., 
2022). While the underlying mechanisms of these cognitive effects remain to be fully elucidated, 
some evidence suggests that they may be mediated, at least in part, by probiotic-induced 
modulation of metabolic function. Several meta-analyses of randomised controlled trials have 
reported that probiotic supplementation significantly improves glycaemic control in both clinical 
and sub-clinical populations (Nikbakht et al., 2016; Ruan et al., 2015). These effects are thought 
to arise from multiple mechanisms, including modulation of the gut-microbiome, strengthening of 
intestinal and immune barriers, reduction of IR and oxidative stress, and regulation of metabolic 
enzyme activity (Shen et al., 2024). Taken together, these findings suggest that probiotics may 
exert cognitive effects through their capacity to restore glucose-specific aspects of metabolic 
function.  
  
Typically, research examining the effects of probiotics on both glucose metabolism and 
cognition has focused on the genera Lactobacillus and Bifidobacterium (McCarthy & 
Martirosyan, 2025; Pintarič & Langerholc, 2022). Assessing the therapeutic efficacy of probiotics 
is challenging however, due to strain and dose-specific variations in effects (Torres-Maravilla et 
al., 2022). Among these, Lab4P, a probiotic consortium consisting of five Bifidobacterium and 
Lactobacillus bacterial strains, has been investigated for its potential to exert favourable 
metabolic effects. Randomized controlled trials have found Lab4P supplementation in 
overweight and obese adults significantly reduces body weight, waist circumference, and hip 
circumference compared to placebo (Michael et al., 2020; Michael et al., 2021). The first of 
these studies also reported improvements in participants' overall wellness scores. Animal 
studies further support this, with rats given Lab4 probiotics showing lower systemic inflammation 
and improved lipid profiles, potentially via altered gut bile acid metabolism (Joyce et al., 2014; 
Webberley et al., 2021). Moreover, probiotic strains belonging to the same genera as those 
comprising Lab4P have been shown to improve glycaemic regulation in prediabetic adults by 
enhancing lipid metabolism, reducing inflammation, alleviating oxidative stress and stimulating 
the secretion of GLP-1 in the body (Abolbaghaei et al., 2018; Li et al., 2022). Given the 
established links between metabolic dysfunction, inflammation, and cognitive decline, these 
findings suggest that Lab4P may offer a strategy not only for improving metabolic health but 
also for supporting cognitive function.   
  
Indeed, preclinical studies suggest that Lab4P may exert direct neuroprotective effects. In 
animal models, Lab4P supplementation preserved hippocampal dendritic spine density, and 
prevented learning and memory deficits induced by a high-fat diet, indicating protection against 
synaptic degeneration linked to metabolic dysfunction (Webberley et al., 2021). These structural 
benefits were accompanied by suppressed expression of pro-inflammatory cytokines, and 
preservation of anti-inflammatory signalling, within the hippocampus. Further, in vitro, Lab4P-
derived metabolites protected human cholinergic neuronal cells from β-amyloid–induced 
cytotoxicity and significantly reduced IL-6 expression (Webberley et al., 2021). These findings 
suggest that Lab4P may mitigate cognitive decline by preserving synaptic integrity and 
dampening neuroinflammation in metabolically vulnerable states. The precise mechanisms 
underlying these effects remain to be fully elucidated, however, Lab4P harbours genes involved 
in the production of short-chain fatty acids (SCFAs), which are known to modulate systemic 
inflammation and may exert anti-inflammatory effects within the brain (Li et al., 2018; Vinolo et 
al., 2011). Further, microbiota-derived SCFAs are thought to mitigate hyperglycaemia-mediated 
oxidative stress and inflammatory response and appear to preserve the integrity of 

 
 
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cerebrovascular endothelial cells in the presence of ROS and AGEs (Huang et al., 2017; 
Kassan et al., 2023; Yan et al., 2022). This is particularly relevant in the context of IGM, where 
such mechanisms are associated with impaired NVU coupling and cognitive decline. Together, 
the evidence suggests that Lab4P may exert protective effects on cerebral function in 
metabolically vulnerable states by modulating key pathways implicated in cognitive impairment. 
While direct evidence of Lab4P’s effects on cognition in humans is limited, its demonstrated 
ability to reduce systemic inflammation and improve metabolic parameters indicates potential 
relevance.  
 
2. RATIONALE  
IGM is a precursor to T2DM and other metabolic diseases. Increasing evidence also links IGM 
to early cognitive decline, likely mediated by insulin resistance, chronic inflammation, and 
endothelial dysfunction. Young adults with IGM represent a critical intervention group, as early 
metabolic dysregulation significantly increases the risk of progression to more severe 
cardiometabolic and neurological disorders. Intervening at this early stage may offer a valuable 
opportunity to prevent or even reverse these effects before they become irreversible. 
 
Current therapeutic strategies for metabolic dysfunction primarily involve either lifestyle 
modification or pharmacological treatment (Blüher et al., 2023). These strategies have found 
concurrent enhancements in glycaemic regulation and cognitive performance (Fontana et al., 
2021; Komleva et al., 2021). Lifestyle interventions, such as dietary modification and increased 
physical activity, can be effective; however, long-term adherence remains a significant 
challenge (Perone et al., 2024). Pharmacological interventions that improve glucose metabolism 
parameters have also been associated with improving weight management, glycaemic control, 
and, in some cases, cognitive function (Hui et al., 2025; Vosoughi et al., 2022). For example, a 
recent study examining liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist that 
enhances insulin secretion and improves glycaemic control, also improved associative learning 
in young obese individuals (Hanssen et al., 2023). Nonetheless, the broader use of GLP-1 
receptor agonist, and similar medication is constrained by high cost, potential adverse effects, 
and the frequent recurrence of symptoms upon treatment discontinuation (Sikirica et al., 2017). 
These limitations highlight the need for alternative approaches that are effective, better 
tolerated, and more sustainable for long-term management of metabolic dysfunction. 
 
Probiotics offer a promising alternative or adjunctive approach. They are generally well 
tolerated, with fewer side effects than pharmacological agents, and may promote lasting 
metabolic improvements through modulation of the gut microbiome (Torres et al., 2024). The 
Lab4P probiotic consortium has shown encouraging results in improving metabolic markers and 
reducing inflammation in overweight adults, and preclinical studies suggest additional 
neuroprotective effects. However, its potential to improve cognitive function in humans with IGM 
has not yet been evaluated. Clinical research is needed to establish whether Lab4P can 
mitigate the cognitive and behavioural impairments associated with metabolic dysfunction.  
 
This trial will evaluate whether Lab4P supplementation, compared to placebo, can enhance 
cognitive performance, and metabolic health in overweight young adults with IGM, aiming to 
establish it as a potential non-pharmacological strategy to mitigate long-term neurocognitive and 
metabolic decline. The inclusion of a placebo control is scientifically and ethically justified in this 
context. Given the absence of an established probiotic intervention for IGM, a placebo provides 

 
 
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an appropriate comparator, enabling a rigorous evaluation of efficacy and reducing the risk of 
bias due to expectancy effects or non-specific changes. 
 
2.1 Assessment and management of risk 
This trial involves the use of the probiotic Lab4P or a matching placebo. The safety profile of 
Lab4P is well established, with its constituent strains used in commercially available products. 
The anticipated risks are minimal and consistent with those seen in everyday probiotic use. 
No procedures in this study exceed the level of standard care. Stool and blood sample 
collection, DEXA scans, and blood pressure measurements are routine clinical and research 
assessments and will be performed by trained professionals using approved SOPs. Participants 
will be fully informed of potential side effects and provided with access to the research team 
should concerns arise. 
The table below outlines the potential risks associated with each intervention component and 
the corresponding risk mitigation strategies: 
Intervention 
Potential Risk 
Risk Management 
Oral dose of Lab4P 
probiotic 
Allergic reaction 
Individuals with a known allergy to 
probiotics or any component of the 
supplement will be excluded from 
participation. Participants will also be 
monitored for any adverse events 
throughout the trial. 
Oral dose of Lab4P 
probiotic 
Gastrointestinal 
symptoms 
Participants will be informed of the 
possibility of temporary gastrointestinal 
symptoms. They will have access to the 
PI to report symptoms or seek advice at 
any time. Ongoing symptoms will be 
monitored and managed according to 
clinical guidelines. 
Oral dose of placebo 
containing 
microcrystalline 
cellulose  
Allergic reaction 
Microcrystalline cellulose is considered to 
have a very low allergenic potential. 
Nonetheless, participants will be informed 
of this minimal risk. Any participant with a 
known sensitivity to excipients will be 
excluded. 
 
 
3 OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS 
Baseline is defined as the assessments conducted prior to randomisation (Weeks –1 and 0). 
Follow-up assessments will take place at Weeks 6, 11, and 12 following randomisation. 
 

 
 
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3.1 Primary objective 
The primary objective of this trial is to evaluate the effect of once-daily Lab4P probiotic 
supplementation (5 × 10¹⁰ CFU) versus placebo on memory performance over a 12-week 
intervention period in overweight adults with IGT. 
 
3.2 Secondary objectives 
To evaluate the effect of Lab4P versus placebo on the following secondary outcomes in 
overweight adults with IGT:  
• 
Performance across five computerized cognitive tasks measuring memory, executive 
function, and psychomotor speed 
• 
Cardiometabolic function 
• 
Peripheral and central vascular function 
• 
Inflammatory status 
• 
Anthropometry  
• 
Body composition 
• 
Fecal microbiota community composition, dispersion and abundance of individual 
microbial taxa 
• 
Subjective sleep quality 
 
3.2.1 Secondary Safety Objectives 
To assess the tolerability and safety profile of Lab4P over 3 months. 
 
3.3 Primary endpoint/outcome 
The primary endpoint is the change from baseline to week 12 in mean verbal memory 
performance, assessed by accuracy on the Cambridge Neuropsychological Test Automated 
Battery (CANTAB) verbal recognition memory (VRM) task.  
 
3.4 Secondary endpoints/outcomes 
 
3.4.1 Cognitive Performance Endpoints  
Changes from baseline to Week 6 and Week 12 in performance on the following computerized 
CANTAB cognitive tasks:  
• 
Paired Associates Learning (PAL) task, assessing visual episodic memory and 
associative learning.  
• 
Spatial Working Memory (SWM) task, assessing visuospatial working memory and 
strategy use.  
• 
Stop Signal Task (SST), assessing response inhibition and impulse control.  
• 
Intra-Extra Dimensional Set Shift (IED) task, assessing cognitive flexibility and 
attentional set-shifting.  
• 
Five Choice Reaction Time (RTI) task, assessing processing speed, psychomotor 
response, and attention.  
 
3.4.2 Sleep Quality Endpoints  
Change from baseline to Week 6 and Week 12 in subjective sleep quality, assessed using 
Pittsburgh Sleep Quality Index (PSQI) component scores. PSQI components include:  

 
 
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− 
Global Sleep Quality Score 
− 
Sleep Latency  
− 
Sleep Duration  
− 
Habitual Sleep Efficiency  
− 
Sleep Disturbances  
− 
Use of Sleep Medication  
− 
Daytime Dysfunction  
 
3.4.3 Cardiometabolic Endpoints 
Change from baseline to Week 12 in:  
− Capillary blood glucose (BG) area under the curve (AUC) during a 2-hour OGTT.  
− HbA1c 
− Fasting BG Level 
− Systolic blood pressure  
− Diastolic blood pressure 
− 
Total cholesterol  
− 
LDL-C  
− 
HDL-C  
− 
Triglycerides  
 
3.4.4 Peripheral and Central Vascular Endpoints 
Change from baseline to Week 12 in: 
− Cerebrovascular reactivity (CVR) of the middle cerebral artery 
− Flow-mediated dilation (FMD) of the brachial artery 
 
3.4.5 Inflammatory Endpoints  
Change from baseline to Week 12 in plasma concentrations of inflammatory cytokines, 
including:  
− 
Interleukin-6 (IL-6)  
− 
Tumor necrosis factor-alpha (TNF-α)  
 
3.4.6 Platelet Activity Endpoints 
 Change from baseline to Week 12 in circulating markers of platelet activity, assessed using 
validated laboratory methods. 
3.4.7 Anthropometric Endpoints  
Change from baseline to week 12 in:  
− 
Body Mass Index (BMI)   
− 
Waist-to-hip ratio.  
 
3.4.8 Body Composition Endpoints  
Change from baseline to week 12 in:  
− 
Total lean mass (TLM)  

 
 
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− 
Total fat mass (TFM)  
− 
Percent body fat.  
 
3.4.9. Gut Microbiome Diversity Endpoints  
Change from baseline to week 12 in:   
− Gut microbiota community composition and diversity   
− 
Gut microbiota community dispersion 
− 
The relative abundance of individual microbial taxa  
 
3.4.10 Secondary Safety Endpoints  
− Proportion of participants with ≥80% adherence to Lab4P, as determined by pill count at 
the Week 12 visit. 
− Number and proportion of participants reporting gastrointestinal (GI)-related adverse 
events (AEs), including but not limited to nausea, diarrhea, bloating, constipation, and 
abdominal discomfort, as captured through a recall diary. 
 
3.5 Table of endpoints/outcomes 
Objectives 
Outcome Measures  
Timepoint(s) of 
evaluation of this 
outcome measure 
Primary Objective 
To evaluate the effect of once-
daily Lab4P probiotic 
supplementation (5 × 10¹⁰ CFU) 
versus placebo on memory 
performance over a 12-week 
intervention period in overweight 
adults with IGT 
Accuracy score on the CANTAB 
Verbal Recognition Memory (VRM) 
Task.  
Week 0, week 6 and week 
12. 
Secondary Objectives 
Evaluate the effect of Lab4P 
versus placebo on memory, 
executive function, and 
psychomotor speed 
performance. 
 
Paired Associates Learning (PAL) 
task performance. 
Spatial Working Memory (SWM) task 
performance. 
Stop Signal Task (SST) performance. 
Intra-Extra Dimensional Set Shift 
(IED) task performance. 
Five Choice Reaction Time (RTI) 
task performance. 
Week 0, week 6 and week 
12. 
 
Evaluate the effect of Lab4P 
versus placebo on Subjective 
sleep quality 
The following measures of the PSQI:  
Global Sleep Quality Score 
Sleep Latency  
Sleep Duration  
Habitual Sleep Efficiency  
Week 0, week 6 and week 
12. 

 
 
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Sleep Disturbances  
Use of Sleep Medication  
Daytime Dysfunction 
Evaluate the effect of Lab4P 
versus placebo on 
cardiometabolic function 
Capillary BG AUC during a 2-hour 
oral glucose tolerance test (OGTT).  
HbA1c 
Fasting BP Level 
Systolic blood pressure  
Diastolic blood pressure 
Total cholesterol  
LDL-C  
HDL-C  
Triglycerides 
Week –1, week 6 and week 
12.  
Evaluate the effect of Lab4P 
versus placebo on inflammatory 
status 
Circulating plasma Interleukin-6 (IL-
6)  
Circulating plasma Tumor necrosis 
factor-alpha (TNF-α) 
 
Week -1 and week 12. 
 
Evaluate the effect of Lab4P 
versus placebo on platelet 
activity 
Circulating markers of platelet activity 
Baseline, Week 6 and 
Week 12 
Evaluate the effect of Lab4P 
versus placebo on 
anthropometry  
BMI   
Waist-to-hip ratio. 
Week –1, Week 6 and 
week 12. 
 
Evaluate the effect of Lab4P 
versus placebo on body 
composition 
Total lean mass (TLM)  
Total fat mass (TFM)  
Percent body fat. 
Week 0 and week 12. 
 
Evaluate the effect of Lab4P 
versus placebo on peripheral 
and central vascular function 
Cerebrovascular reactivity (CVR) of 
the middle cerebral artery 
Flow-mediated dilation (FMD) of the 
brachial artery 
Week 0 and week 12.  
Evaluate the effect of Lab4P 
versus placebo on fecal 
microbiota community 
composition and community 
dispersion 
The relative abundance of individual 
microbial taxa   
Gut microbiota community 
composition    
Gut microbiota community dispersion   
Week 0 and week 12.  
Secondary Safety Objectives 
Assess the tolerability and 
safety profile of Lab4P over 3 
months. 
 
Proportion of participants with ≥80% 
adherence to Lab4P, as determined 
by pill count at the Week 12 visit. 
 
Number and proportion of 
participants reporting physical 
symptoms and GI-related AEs, 
Weeks 0, 1, 2, 3, 4, 5, 6, 7, 
8, 9, 10, 11, 12 

 
 
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including but not limited to nausea, 
diarrhea, bloating, constipation, and 
abdominal discomfort, as captured 
through a weekly recall diary. 
 
 
4. TRIAL DESIGN 
This study is a 12-week, randomized, double-blind, placebo-controlled, two-arm, parallel-group, 
single-centre superiority trial. It is designed to evaluate the effect of once-daily Lab4P probiotic 
supplementation (5 × 10¹⁰ CFU) compared to placebo on cognitive performance and sleep 
quality in overweight adults with IGT. 
Participants will be randomly assigned in a 1:1 ratio to receive either the probiotic or a matching 
placebo for the duration of the intervention. Study visits and assessments will occur at five time 
points: Screening (Week –1), Baseline (Week 0), Mid-Intervention (Week 6), Pre-Final 
Assessment (Week 11), and End-of-Intervention (Week 12). GI-related adverse events will be 
recorded weekly throughout the intervention using participant diaries. Outcome measures will 
be collected at each visit as appropriate, with primary endpoints assessed at Week 12. A 
summary of the visit schedule is provided in Section 4: Diagrammatic Schedule of Visits and 
Assessments. 
 
 
5 TRIAL SETTING 
This is a single-centre trial, and no additional sites are involved in recruitment, treatment, or 
follow-up. All experimental trial activities will be conducted at a single site: the Carnegie School 
of Sport at the Leeds Beckett University (LBU) Headingley Campus. The Carnegie School of 
Sport provides the necessary facilities and specialist staff required to run the intervention and 
data collection activities. All trial interventions will be administered by appropriately qualified 
personnel, meeting any professional eligibility criteria relevant to the intervention. Participants 
will be recruited from the local population, primarily through community and university channels, 
as the trial is not targeting a clinical patient population.  
 
 
6 PARTICIPANT ELIGIBILITY CRITERIA 
The study population will consist of otherwise healthy adults aged 18 to 40 years, with a Body 
Mass Index (BMI) classified as overweight (25.0–29.9 kg/m²; Weir & Jan, 2023), and who have 
IGT. IGT will be defined by a total area under the curve (AUC) for capillary blood glucose ≥290 
mg·h/dL during a 2-hour OGTT (Sakaguchi et al., 2015). Participants must self-report good 
general health. This population was selected to evaluate early-stage metabolic dysfunction in a 
relatively homogeneous cohort and to improve the generalisability of findings to similar at-risk 
individuals. 
 
6.1 Inclusion criteria 
Participants are eligible for enrolment in the study if all the following criteria are met:  
− 
Provision of written informed consent prior to any study-related procedures.  
− 
Aged 18 to 40 years, inclusive, at the time of consent.  

 
 
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− 
BMI between 25.0 and 29.9 kg/m².  
− 
Self-reported good general health with no chronic medical conditions requiring ongoing 
treatment.  
− 
Self-reported normal sleep patterns with no history of diagnosed sleep disorders.  
− 
Willing and able to comply with study procedures, including fasting and undergoing the 
OGTT. 
− 
Non-smoker at time of screening, with no regular tobacco use in the last 6 months. 
− 
Proficient in English equivalent to a native speaker. 
 
6.2 Exclusion criteria 
Participants will be excluded from the study if any of the following criteria apply: 
− 
Current or past diagnosis of any form of diabetes mellitus. 
− Fasting plasma glucose ≤3.9 and ≥7.0 mmol/L at screening. 
− 
History of bariatric surgery (e.g., gastric bypass, sleeve gastrectomy) or other 
gastrointestinal procedures that may influence glucose absorption or metabolism. 
− 
Major surgery, significant trauma, systemic infection, or myocardial infarction within six 
weeks prior to screening. 
− 
History of kidney or liver disease. 
− 
History of Low/High Blood Pressure or heart disease 
− History of any health conditions that would effect food metabolism including the 
following: food allergies, kidney disease, liver disease and/or gastrointestinal diseases 
(e.g. irritable bowel syndrome, peptic ulcers)?   
− 
Current or recent (within 4 weeks) use of medications known to affect glucose 
metabolism or agents known to alter gut microbiota composition or probiotic efficacy (as 
defined in Section 8.6). 
− 
History of psychiatric illness, including anxiety or depression. 
− 
Current use of prescription or over-the-counter medications, excluding hormonal 
contraception. 
− 
Participation in night shift work within 30 days prior to screening or planned night shift 
work at any time during the study. 
− 
Pregnancy, breastfeeding, or planning to become pregnant during the study period. 
− 
Known allergy or hypersensitivity to any component of the investigational product or 
placebo. 
 
 
7 TRIAL PROCEDURES  
All procedures and assessments outlined below are aligned with the study objectives defined in 
Section 3 and are scheduled as per the Schedule of Activities (Appendix A).   
 
7.1 Recruitment 
Participants will be recruited from the general population within the local community, including 
both university-affiliated and non-university-affiliated individuals. Recruitment will target adults 
aged 18 to 40 years who meet the eligibility criteria defined in Section 6. While the student body 
at LBU represents a key recruitment pool, the study is open to all eligible individuals within the 
local area. 

 
 
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Recruitment will be conducted using a combination of digital and in-person strategies. These will 
include: 
− 
Email invitations circulated via university mailing lists 
− 
Physical posters placed in high-traffic locations across university campuses and 
community venues 
− 
Social media advertisements posted through institutional and local accounts 
− 
In-class announcements (with prior approval from academic staff) 
− 
Online postings on research participation websites and community bulletin boards 
 
Recruitment materials requiring gatekeeper access (e.g., private social media groups) will only 
be shared after obtaining permission from the relevant gatekeepers. All recruitment materials 
will clearly communicate the study’s purpose, voluntary nature, participant requirements, and 
the key inclusion and exclusion criteria. Contact information for the research team will be 
provided. No covert or deceptive recruitment methods will be used. 
Where members of the research team hold teaching or supervisory responsibilities, recruitment 
will be conducted entirely outside any academic assessment context to mitigate perceived 
coercion. Recruitment will continue on a rolling basis until the required sample size is achieved, 
as outlined in Section 10.2. 
 
7.1.1 Participant identification 
Potential participants will be recruited through self-referral in response to study advertisements. 
These materials will direct individuals to a secure Qualtrics link, accessible via QR code or by 
contacting the PI, where they can view the Participant Information Sheet (PIS) and complete a 
brief online pre-screening questionnaire. This pre-screening form will determine initial eligibility 
before individuals are invited to a formal screening visit. 
 
This questionnaire will include mandatory questions with embedded logic checks. The form will 
screen for age (18–40 years), general health status, diabetes history, recent major medical 
events, pregnancy status, sleep patterns, shift work, and willingness to comply with pre-trial 
requirements. Participants will self-report height and weight, from which BMI will be calculated 
(target range: 25.0–29.9 kg/m²). Medication and supplement use will also be reported to identify 
any exclusions related to substances that may interfere with study outcomes (see Section 10.2). 
 
Based on predefined eligibility criteria, the Qualtrics platform will automatically determine 
eligibility and notify participants of their status. Those who meet the criteria will be redirected to 
an online scheduling system (Google Calendar) to book their first laboratory appointment. 
Appointment availability will be managed in real time by the research team to ensure 
appropriate scheduling. The pre-screening questionnaire must be completed no more than one 
month prior to the scheduled screening visit to ensure information remains current and accurate. 
 
To support transparent reporting in line with CONSORT guidelines, anonymised data will be 
collected on all individuals who complete pre-screening but are not enrolled. This will include 
age, gender, ethnicity, eligibility status (eligible/ineligible), and, where applicable, the reason for 
ineligibility or reason for declining participation. These data will be used solely for reporting 
recruitment flow and assessing generalisability and will not be linked to personally identifiable 
information. 

 
 
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7.1.2 Screening Procedures 
Participants who meet the initial pre-screening criteria will be invited to attend a screening visit 
(Visit 1), which must occur within 14 days prior to the baseline visit (Visit 2). The purpose of this 
visit is to confirm eligibility through defined clinical, anthropometric, and laboratory assessments, 
and to obtain written informed consent before initiating any study procedures. 
Following consent, the study team will verify adherence to pre-trial requirements (see Table 2). 
Anthropometric measurements will be taken, including height, weight, and waist and hip 
circumference, for calculation of BMI. Participants will then have their fasting BG tested using a 
validated CE-marked glucometer.  
 
Table 2: Pre-trial requirements.  
Requirement 
Details  
Timeframe Before 
visit 
Alcohol restriction 
No consumption of alcoholic beverages 
24 hours 
Fasting and beverage 
restriction 
No food or caloric beverages. Water is 
permitted. 
10 hours 
Smoking restriction 
No smoking or use of nicotine products 
10 hours 
Vigorous physical activity 
restriction 
No vigorous exercise (e.g., running, high-
intensity cycling, competitive sports). 
10 hours 
 
To be eligible for inclusion, participants must meet all of the following criteria at screening: 
− BMI between 25.0 and 29.9 kg/m² 
− Fasting capillary glucose < 7.0 mmol/L 
− 
Confirm compliance with all pre-trial requirements 
 
Eligibility will be reviewed and confirmed prior to baseline assessments and randomisation. 
Participants who do not meet the eligibility criteria will be classified as screen failures. However, 
rescreening may be allowed once, at the discretion of the study team, if the ineligibility is due to 
a temporary or correctable factor (e.g. recent supplement use or minor BMI deviation). All 
rescreening decisions and rationale will be documented in the case report form (CRF). 
Participants who complete the screening visit will be issued a Fe-Col® stool collection kit with 
written instructions. They will be asked to collect the sample at home within 24 hours prior to the 
baseline visit and return it in the provided container. 
 
7.1.3 Cognitive Task Familiarization 
During the Screening visit (week –1) participants will complete a cognitive task familiarization 
session involving the Motor Screening Task (MOT) from the Cambridge Neuropsychological 
Test Automated Battery (CANTAB; Robbins et al., 1998). This serves to ensure that participants 
are comfortable with the CANTAB system prior to the main cognitive battery. The MOT provides 
a baseline measure of sensorimotor coordination, visual comprehension, and motor 
responsiveness, and helps identify impairments that may compromise the validity of subsequent 
cognitive data (Soares et al., 2014). Although not a direct measure of cognition, it ensures 
participants possess the necessary motor and perceptual skills to engage reliably with tasks. 
This task is expected to take around 2 minutes to complete. Task outcomes are outlined in table 

 
 
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3.  
 
Table 3: Motor Screening Task Outcomes 
Outcome 
Measures 
Description 
Unit of 
Measurement 
Mean Latency 
The average time (in milliseconds) between 
stimulus onset and participant response across 
ten trials.   
Milliseconds (ms) 
Mean Error 
The average spatial deviation between the center 
of the target and the point of contact registered on 
the screen.   
Millimeters (mm) 
 
7.1.3 Payment  
Participants will receive a voucher for £100 upon completion of the study to recognize their time 
and contribution. This amount is intended to compensate for the inconvenience of attending five 
in-person visits, completing assessments, and adhering to study procedures over the 12-week 
period. The payment will be made in full at the final study visit (Week 12), contingent on 
completion of all required visits and procedures. 
 
This reimbursement is not intended as an incentive to participate but rather as fair 
compensation for time and effort, in accordance with HRA guidance on payments in research 
(HRA, 2020). No additional travel expenses will be reimbursed, as all study visits take place 
within a single site easily accessible to participants through public and university transport links. 
 
7.2 Consent  
Participation in this study is entirely voluntary. Informed consent will be obtained in accordance 
with the principles of Good Clinical Practice (GCP), the Declaration of Helsinki, and applicable 
regulatory and institutional standards. The PI retains overall responsibility for ensuring that the 
informed consent process is conducted appropriately at the study site. Any member of the 
research team delegated to take consent will be trained, competent, and authorised to do so. 
 
All prospective participants will be provided with a LREC approved PIS at least 24 hours before 
the screening visit. The PIS will clearly outline the purpose, procedures, duration, potential risks 
and benefits of the study, the voluntary nature of participation, the right to withdraw at any time 
without penalty, and data confidentiality protections. It will also state how participants may 
contact the research team for additional information. 
 
At the screening visit (Visit 1), individuals will be given the opportunity to ask questions and 
discuss the study with a member of the research team before giving consent. Written informed 
consent will be obtained prior to any study-specific procedures, including the collection of 
identifiable data or the initiation of screening assessments. 
 
The process of obtaining consent will be documented in the study records. A signed copy of the 
informed consent form (ICF) will be given to the participant, and the original will be retained 
securely with the study documentation. Where appropriate, additional consent will be obtained 
to allow for the use of data and samples collected prior to withdrawal, as outlined in the consent 

 
 
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materials. 
 
Participants will be assumed to have capacity to consent unless evidence indicates otherwise. 
The research team will ensure that individuals are capable of understanding the study and 
making an informed decision at the time of consent. Capacity will be assessed informally by the 
consenting researcher in line with ethical guidelines. The study will exclude individuals who are 
unable to provide informed consent for themselves. This trial does not involve the enrolment of 
children, young people under 18 years of age, or adults lacking mental capacity. 
 
The study does not involve any procedures requiring translation services; however, if a 
participant requests written materials in another language, the research team will make 
reasonable efforts to provide translated versions of key documents (e.g. PIS and ICF, subject to 
availability. Due to funding limitations, the hiring of professional verbal interpreters is not 
included in the study budget, and verbal translation services cannot be provided by the research 
team. 
 
Should any new information arise during the study that may influence a participant’s willingness 
to continue, the PI will ensure that this is communicated promptly and that re-consent is 
obtained using updated, REC-approved materials. 
 
7.2.1 Additional consent provisions for collection and use of participant data and biological 
specimens in ancillary studies 
Participants will be asked to provide additional, optional consent for the storage and future use 
of their blood and stool samples beyond the main trial. These samples may be used in ancillary 
studies related to metabolic health, probiotic interventions, or gut microbiome research, 
including projects conducted by universities, NHS or non-profit research institutions, or 
commercial research partners, both in the UK and internationally. 
 
Participation in ancillary research is entirely optional and not a requirement for inclusion in the 
main trial. Participants may choose whether to allow the use of their biological samples for: 
− Specified ancillary studies related to the current trial 
− Future ethically approved research that may be unrelated to the primary study condition 
− Submission to an approved research tissue bank for long-term storage and broader 
research use 
 
Participants will indicate their preferences on the ICF. All samples used for ancillary research 
will be anonymised (labelled only with a unique participant ID) and handled in accordance with 
the Human Tissue Act (HTA) 2004 and UK GDPR. If a participant consents to future use but 
later decides to withdraw from ancillary research, they may do so at any time by contacting the 
study team. Upon withdrawal, any stored identifiable samples that have not yet been used will 
be securely destroyed. However, data or results already generated from those samples may not 
be retractable due to anonymisation. 
 
Participants will not receive individual results from ancillary research, as these analyses are 
exploratory and not designed for clinical interpretation. Participants will also not be contacted 
regarding findings unless they have given separate consent to be re-contacted for future 

 
 
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studies. 
 
7.3 The randomisation scheme 
Participants will be randomly assigned to either the probiotic intervention group or the placebo 
group with equal allocation (1:1). Randomisation will be performed at the point of manufacture 
by Cultech, the UK manufacturer of Lab4P, using a web based randomisation platform 
(www.sealedenvelope.com), employing permuted block sizes of size 2 to maintain allocation 
balance.  
Each product container will be labelled with a unique investigational product (IP) number 
corresponding to the randomisation sequence.  
 
7.3.1 Method of implementing the randomisation/allocation sequence 
Each participant will be assigned a unique IP number at the point of intervention dispensing. 
These IP numbers correspond to the pre-generated randomisation list and are used to track 
product assignment throughout the trial.  
Study personal will allocate participants the next sequential IP number from the pre-labelled 
containers, without access to the underlying treatment group. The randomisation sequence will 
be stored in a password protected excel file, permitting individual level unblinding if clinically 
required. The password will be held by the primary trial supervisor, Professor Lauren Owen and 
provided in a security sealed envelope labelled with the trial identifier. This envelope will be 
retained securely and returned unopened at the end of the study to confirm that blinding was 
maintained. Professor Owen is not involved in participant-facing procedures or site-based trial 
activities.  
 
The allocation sequence itself will not be accessible to site staff at any point. IP numbers will be 
recorded in the trial master file and case report forms to ensure traceability.  
 
There is no participant registration or randomisation procedure at the study site, as allocation is 
fully managed in advance of product delivery. All trial documentation and data capture systems 
will reference participants by their IP number, ensuring treatment concealment is maintained at 
every stage.  
 
7.4 Blinding 
This trial will implement a blinded design to minimise bias in the delivery, assessment, and 
analysis of the intervention. The investigational product and the placebo will be identical in 
appearance, packaging, and flavour to ensure full comparability and maintain blinding integrity. 
Both products will be provided in matching containers and administered using the same 
instructions for use. The following individuals will be blinded to participant allocation throughout 
the trial: 
− Trial participants 
− 
Research personnel involved in data collection, intervention delivery, and outcome 
assessment 
The project supervisor, who holds the password to the randomisation file, will remain unblinded 
but will not be involved in any site-based procedures, participant interaction, or outcome 
assessment. If operational interaction between blinded and unblinded personnel is required, 
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matters. 
 
Unblinding of individual participants will occur only if necessary for clinical safety and only 
following documented approval by the project supervisor. In such cases, the unblinding process 
will be clearly documented and reported. Final unblinding of all participants and treatment 
groups will take place only after the analysis dataset has been locked and signed off by the 
project supervisor. The trial does not involve any procedures, tests, or equipment that could 
inadvertently reveal group allocation to participants or blinded researchers. 
 
7.5 Emergency Unblinding 
Unblinding may be undertaken only in the case of a valid medical emergency, such as a serious 
or unexpected adverse event, where it is deemed essential to know the participant’s treatment 
allocation in order to provide appropriate clinical care. The decision to unblind must always 
prioritise participant safety, in line with the Declaration of Helsinki and ICH-GCP (Section 4.3). 
Wherever possible, the remainder of the research team will remain blinded. 
 
7.5.1 Code Break Access and Control 
The randomisation code is held securely by Professor Lauren Owen, and access is restricted to 
designated personnel not involved in trial assessments or analysis. The code break mechanism 
will be managed through a secure password-protected database, which permit individual-level 
unblinding only. The unblinding mechanism will be designed to ensure that only the treatment 
allocation of the individual concerned is revealed. Under no circumstances will unblinding of the 
entire trial or access to the full randomisation list be permitted. Unblinding will only be allowed in 
the case of medical emergency.  
 
7.5.2 Emergency Unblinding Procedure 
In the event of a medical emergency, a formal request for unblinding will be made by the PI. The 
code will be accessed by the authorised holder, and the allocation disclosed only to the clinician 
responsible for the participant’s care. The participant will not be informed of their allocation 
unless medically necessary. The code break procedure will allow rapid unblinding, as required 
under ICH GCP 5.13.4. The reason for unblinding, along with the date, time, and signature of 
the individual who performed the unblinding, must be documented in the CRF and the trial 
master file. 
 
7.5.3 Documentation and Notification 
The following documentation and notifications are required following any unblinding: 
− The date, time, and reason for unblinding will be recorded in the participant’s individual 
CRF, and the site file. 
− The PI will notify the project supervisor in writing as soon as possible, including a 
summary of the event and justification for unblinding. 
− The event will be included in the final trial report and, if applicable, in the statistical 
analysis documentation. 
− The PI will notify the LREC and other relevant regulatory bodies if the unblinding is 
associated with a serious adverse reaction or significant protocol deviation. 
− SUSARs are required to be reported unblinded, as per regulatory requirements. 
 

 
 
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7.6 Baseline Data 
All baseline data will be collected during the screening visit (week –1) following screening 
confirmation, and the baseline visit (week 0), prior to randomisation. These measures are 
essential for characterising the study population and for evaluating primary and secondary 
outcomes. Descriptions of all measures are presented in section 8.  
 
7.6.1 Participant Reported Data 
The following categories of participant reported data will be collected:  
− 
Subjective sleep Quality (Week 0) 
− Demographics and lifestyle (Week 0) 
− Dietary Habits (Week 0) 
− Physical symptoms and adverse events (Week 0) 
 
7.6.2 Cognitive Performance Data 
The following Cognitive performance data categories will be collected at the baseline visit (week 
0):  
− 
Memory task performance (3 tasks) 
− 
Executive function task performance (2 tasks) 
− 
Attention and psychomotor speed task performance (1 task) 
 
7.6.3 Clinical Assessment Data 
The following baseline clinical assessments will be conducted at the screening visit (Week –1): 
− Cardiometabolic data (OGTT BG, HbA1c, Lipid profile, Resting blood pressure) 
− Venous blood draw 
− Anthropometric data (Height, Weight, BMI calculation) 
− Body Composition data (Fat mass, lean mass, percentage fat mass) 
− Vascular function (Cerebrovascular reactivity (CVR) of the middle cerebral artery, Flow-
mediated dilation (FMD) of the brachial artery) 
 
7.7 Trial assessments 
This section outlines the timing and content of all trial assessments after baseline. Assessments 
are organized by visit, with visit windows, methods of data collection, and device/protocol 
references provided. A summary schedule of assessments is included in Table 2. Descriptions 
of all measures are presented in section 8. 
 
7.7.1 Testing Visit 2 (Week 6) ±3 days 
The following self-reported and cognitive measures will be collected via Qualtrics: 
− Cognitive performance (CANTAB tasks as above) 
− Venous blood draw  
− Resting blood pressure 
− Fasting BG 
− 
HbA1c 
− 
Lipid profile 
− Weight 

 
 
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− Height 
− Waist and hip circumference 
− Subjective sleep quality  
− Health events and physical experiences 
 
7.7.2 Testing Visit 3 (Week 11) ±3 days 
Cardiometabolic and anthropometric assessments from the baseline section of the screening 
visit will be repeated to assess intervention effects: 
− Fasting BG 
− OGTT BG (0, 30, 60, 120 minutes) 
− HbA1c 
− Lipid profile 
− Venous blood draw  
− Resting blood pressure 
− Weight 
− Height 
− Waist and hip circumference 
 
7.7.3 Testing Visit 4 (End of Intervention, Week 12) ±3 days 
Final outcome assessments will be conducted, repeating baseline measures: 
− Subjective sleep quality 
− Physical symptoms and adverse events 
− CANTAB cognitive battery 
− Peripheral and central Vascular function (Cerebrovascular reactivity (CVR) of the middle 
cerebral artery, Flow-mediated dilation (FMD) of the brachial artery) 
− 
Body composition (Fat mass, lean mass, percentage fat mass) 
 
These data will serve as endpoint measures for all primary and secondary outcomes. 
 
7.8 Withdrawal criteria  
A participant may be withdrawn from the trial or from treatment with the investigational product 
(IP) under the following circumstances: 
 
7.8.1 Participant-Initiated Withdrawal 
Participants have the right to withdraw consent and discontinue participation in the trial at any 
time, without reprisal or loss of benefits, in accordance with the Declaration of Helsinki (World 
Medical Association, 2014). Upon withdrawal of consent, no further trial-specific assessments 
will be conducted, and no additional data will be collected. However, data collected up to the 
point of withdrawal may be retained and used by the PI unless the participant explicitly 
withdraws consent for data processing. 
 
7.8.2 Investigator-Initiated Withdrawal 
The Investigator may withdraw a participant from the trial or the IP if, in their clinical judgment, 
continued participation poses a risk to the participant’s wellbeing. If this decision is based on an 

 
 
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AE or serious adverse event (SAE), it must be reported in accordance with the protocol's safety 
reporting procedures. 
 
7.8.3 Scope of Withdrawal 
 It will be clearly recorded whether the withdrawal pertains to: 
− 
The entire trial (i.e. no further participation or data collection), 
− 
Withdrawal from treatment with the IP but continuation in trial assessments (e.g. 
follow-up visits), or 
− 
Withdrawal from a specific component of the trial, such as translational research. 
 
7.8.4 Documentation and Follow-Up 
 All instances of withdrawal will be documented in the case report form (CRF), including the 
reason for withdrawal if known. The Investigator will make every effort to conduct appropriate 
follow-up, particularly in relation to unresolved AEs, and to ensure the return of any unused 
investigational product. Follow-up procedures will be carried out as per protocol unless consent 
for follow-up has also been withdrawn. 
 
7.8.5 Investigational Product Discontinuation 
 Participants may be discontinued from receiving the IP under the following circumstances: 
− 
Participant chooses to stop treatment with the IP. 
− 
Investigator determines that continued IP exposure is not in the participant’s best 
interest (e.g. due to AEs or SAEs). In this case, the event will be reported 
appropriately. 
Participants who discontinue the IP will not continue with the trial and will be withdrawn from 
further scheduled visits and assessments. Temporary disruptions to IP administration do not 
constitute formal discontinuation and will be handled according to specific guidance in the 
protocol. 
 
7.8.6 Replacement of Withdrawn Participants 
 Participants who discontinue prior to trial completion will not be replaced. 
 
7.8.7 Trial Termination Criteria 
 The trial may be prematurely terminated for reasons including, but not limited to: 
− 
Decision by a regulatory authority, LREC, or supervisory team due to safety 
concerns or feasibility issues, 
− 
Project supervisor decision based on emerging data or other significant 
considerations. 
In the event of early termination or suspension, the Investigator will promptly inform all trial 
participants and study staff. All trial materials will be collected, and the case report forms will be 
completed to the extent possible. 
 
7.9 End of trial 
The end of the trial will occur within 28 days (±7 days) of the 64th participant completing their 
final follow-up visit at Week 12. This period allows for any final data collection, safety 
monitoring, and administrative closure in line with GCP requirements. 
 

 
 
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8 MATERIALS AND MEASURES 
 
8.1 Participant reported data  
All participant-reported data will be collected electronically using the Qualtrics survey platform. 
This method ensures secure, standardized administration and allows for real-time data capture, 
minimization of missing responses through enforced validation rules, and improved data quality 
through automated logic and branching. The platform also facilitates remote completion when 
applicable.  
 
8.1.1 Health Events and Physical Experiences 
To evaluate the impact of Lab4P on commonly occurring gastrointestinal and systemic 
symptoms, participants will be asked to complete a structured, self-administered questionnaire, 
adapted from the Gastrointestinal Symptom Rating Scale (GSRS; Kulich et al., 2008). This 
questionnaire is designed to capture the frequency and severity of commonly reported 
gastrointestinal symptoms, as well as other physical health experiences. 
Participants will be asked to review a list of health-related experiences and, for each, report: 
The number of days they experienced the symptom or event in the past 14 days (0–14), and 
The typical severity of the experience during that time, using a 7-point scale ranging from “No 
discomfort at all” to “Very severe discomfort.” 
 
The questionnaire includes a comprehensive list of gastrointestinal symptoms, such as 
abdominal pain or discomfort, heartburn, acid reflux, nausea, bloating, belching, bowel changes 
(e.g., constipation, diarrhoea, urgency), and sensations of incomplete emptying. 
It also includes other physical health experiences, including sore throat, headache, muscle 
ache, symptoms of common cold, and recent use of oral antibiotics. For participants assigned 
female at birth, additional items include the use of vaginal pessaries or creams, and the 
occurrence of menstruation. 
 
8.1.2 Sleep Quality 
Sleep quality will be evaluated using the Pittsburgh Sleep Quality Index (PSQI), a validated, 
self-administered instrument designed to measure subjective sleep quality and disturbances 
over a 1-month interval (Buysse et al., 1989). The PSQI consists of 19 self-rated items grouped 
into seven component domains:  
− 
Subjective sleep quality  
− 
Sleep latency  
− 
Sleep duration  
− 
Habitual sleep efficiency  
− 
Sleep disturbances  
− 
Use of sleep medication  
− 
Daytime dysfunction  
Each component is scored on a 0–3 scale, with higher scores indicating greater impairment. 
The sum of these component scores yields a global PSQI score ranging from 0 to 21, where 
scores >5 typically indicate poor sleep quality.  
 

 
 
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8.2.3 Demographics and Lifestyle 
The following demographic and lifestyle variables will be collected using a single self-report 
questionnaire composed of validated instruments: 
− 
Sex assigned at birth: Male or Female (self-reported) 
− 
Ethnicity: Categorised according to the most recent UK census classifications (Office for 
National Statistics, 2023) 
− 
Socioeconomic status (SES): Assessed using the National Statistics Socio-economic 
Classification (NS-SEC; Rose & Pevalin, 2003), derived from occupation and education 
data 
− 
Alcohol use: Measured using the Alcohol Use Disorders Identification Test (AUDIT; 
Horváth et al., 2023) 
− 
Physical activity: Measured using the International Physical Activity Questionnaire – 
Short Form (IPAQ-SF; Lee et al., 2011) 
 
8.1.4 Dietary Habits 
Dietary habits will be captured using the EPIC-Norfolk 7-Day Diet Diary (7dDD), a validated, 
structured food diary originally developed from the British Birth Cohort tool (Lentjes et al., 2013). 
Participants will be instructed to record all food and drink consumed over seven consecutive 
days, including weekdays and weekend days, with details on portion sizes, preparation 
methods, and time of intake. Each day is divided into eight sections: seven meal time slots 
covering intake from midnight to midnight, and a final section for recording snacks, additional 
items, and contextual information. 
 
8.2 Cognitive Performance 
Cognitive performance testing will be administered using the Cambridge Neuropsychological 
Test Automated Battery (CANTAB) software, a computerized tool designed to assess several 
cognitive domains (Robbins et al., 1998). CANTAB has been extensively validated and used in 
the assessment of cognitive function across a broad range of neurological and psychiatric 
disorders (Aslam et al., 2018; Janssen et al., 2013). Further, CANTAB tasks have demonstrated 
sensitivity to detecting both cognitive deficits linked to clinical and subclinical metabolic 
dysfunction, as well as the cognitive effects of probiotic interventions (Ahmed et al., 2025; Allen 
et al., 2016; Wild & Musser, 2014). The CANTAB Connect Research software is validated for 
use on all iPad Air devices and is fully compliant with GCP guidelines. The full battery of tasks is 
outlined below. 
 
8.2.1 Memory Tasks 
Task 
Outcome Measures 
Description 
Unit of 
Measurement 
Verbal 
Recognition 
Memory 
(VRM) 
Free Recall Score 
Number of words recalled 
correctly during the free recall 
phase.  
Count (n) 
Immediate 
Number of correctly identified 
Count (n) 

 
 
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Recognition Correct 
target words in the immediate 
recognition phase 
Immediate 
Recognition Errors 
Number of incorrect (distractor) 
selections in the immediate 
recognition phase 
Count (n) 
 
Delayed Recognition 
Correct 
Number of correctly identified 
target words in the delayed 
recognition phase 
Count (n) 
 
Delayed Recognition 
Errors 
Number of incorrect (distractor) 
selections in the delayed 
recognition phase 
Count (n) 
 
Paired 
Associates 
Learning 
(PAL) 
Total Errors Adjusted 
Sum of location errors across 
stages, adjusted for unattempted 
stages due to task termination.  
Count (n) 
Stages Completed 
Number of successfully 
completed stages as task 
difficulty escalates.   
Count (n) 
Trials to Success 
Number of trials required to 
correctly learn all pattern-location 
pairings within a stage. 
Count (n) 
Spatial 
Working 
Memory 
(SWM) 
Between-Search 
Errors 
Number of times a previously 
successful box is revisited in a 
new search.   
Count (n)  
Within-Search Errors 
(count): 
Number of successfully 
completed stages as task 
difficulty escalates. 
Count (n) 
Strategy Score 
Computed based on first box 
selection consistency in 6–8 box 
trials. 
(unitless index) 
 
8.2.2 Executive Function Tasks 
Task 
Outcome Measures 
Description 
Unit of 
Measurement 
Stop Signal 
Task (SST) 
Stop Signal Reaction 
Time (SSRT) 
Estimated latency of stopping an 
already started action 
Milliseconds 
(ms) 
 
Go Reaction Time 
Time from cue onset to response 
in go trials. 
Milliseconds 
(ms) 
Proportion of 
Successful Stops 
Percentage of trials where the 
participant successfully inhibits 
an already started response. 
Percentage (%) 
Intra-Extra 
Dimensional 
Set Shift (IED) 
Total Errors 
Sum of all incorrect responses 
across the task. 
Count (n) 
Stages Completed 
Number of correctly completed 
Count (n) 

 
 
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discrimination stages. 
 
Extra-Dimensional 
Shift Errors 
Errors specifically made during 
the extra-dimensional shift 
phase. 
Count (n) 
 
8.2.3 Attention and psychomotor speed Tasks 
Task 
Outcome Measures 
Description 
Unit of 
Measurement 
Five Choice 
Reaction Time 
(RTI) 
Reaction Time 
Time from stimulus onset to 
button release. 
Milliseconds 
(ms) 
Movement Time 
Time from button release to 
screen touch. 
Milliseconds 
(ms) 
 
Accuracy 
Proportion of correct responses 
across trials. 
Percentage (%) 
 
8.3 Laboratory assessments 
 
8.3.1 Cardiometabolic data 
All of the following data that includes capillary sampling will be performed by trained study 
personnel under the supervision of the Project Lead, following standardised SOPs. Results will 
be recorded directly into the eCRF. 
 
8.3.1.1 Blood Glucose 
Capillary blood will be collected via fingertip lancet (Fisher Scientific). BG measurements will be 
conducted via direct application to single-use glucose test strips inserted into a validated point-
of-care glucometer. Fasting glucose and OGTT responses will be measured using a validated, 
CE-marked glucometer. BG will be assessed at four timepoints during the OGTT: fasting (0 
min), and at 30, 60, and 120 minutes post-glucose ingestion. 
 
8.3.1.2 Glucose Tolerance  
Glucose tolerance will be assessed via the incremental area under the curve (AUC) for capillary 
blood glucose during a standardized 2-hour OGTT. The primary metabolic endpoint will be the 
total AUC for capillary glucose, calculated using a weighted approximation method adapted 
from Sakaguchi et al. (2015). The AUC will be expressed in units of mmol·h/L, with glucose 
concentrations in mmol/L and time in hours. The formula is defined as: 
Capillary Glucose AUC= 
𝐶𝐺(0)+𝐶𝐺(30)×2+𝐶𝐺(60)×3+𝐶𝐺(120)×4
4
 
 
where CG(t) represents the capillary glucose concentration at time t minutes post-glucose load. 
 
8.3.1.3 Glycated Hemoglobin (HbA1c) 
Capillary blood will be collected via fingertip lancet (Fisher Scientific) into micro-collection tubes 
(Abbott Laboratories). HbA1c will be measured from using the Afinion 2 (Abbott). 
 

 
 
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8.3.1.4 Lipid Profile 
Capillary blood will be collected via fingertip lancet (Fisher Scientific) into micro-collection tubes 
(Abbott Laboratories). Lipid profile will be analyzed using the Cholestech LDX analyzer (Abbott). 
The following data will be recorded in the eCRF: 
− 
Total cholesterol 
− 
LDL-C 
− 
HDL-C 
− 
Triglycerides 
 
8.3.1.5 Blood Pressure 
Blood pressure will be measured using a clinically validated, automated oscillometric device 
(Omron). Measurements will be taken after the participant has been seated at rest for 5 
minutes. The following data will be recorded in the eCRF: 
− 
Resting systolic blood pressure (mmHg) 
− 
Resting diastolic blood pressure (mmHg) 
 
8.3.2 Anthropometric data  
Anthropometric measurements will be assessed following ISAK-compliant procedures (Norton, 
2018). The following data will be measured:  
− 
Height (cm) 
− 
Weight (kg) 
− 
Waist circumference (cm) 
− 
Hip circumference (cm) 
Waist circumference is measured at the midpoint between the lower margin of the last palpable 
rib and the top of the iliac crest, while hip circumference is taken at the widest portion of the 
buttocks. Two measurements will be taken at each site, and a third will be performed if the first 
two differ by more than 1 cm; the mean of the closest two values will be recorded.   
From these measurements, BMI will be calculated as weight (kg) divided by height squared 
(m²), and waist-to-hip ratio (WHR) will be calculated as waist circumference divided by hip 
circumference. All anthropometric assessments will be performed by trained research 
personnel, using calibrated equipment to ensure accuracy and reproducibility.   
 
8.3.3 Body Composition data  
Body composition will be assessed using a GE Lunar iDXA scanner (GE HealthCare). Scans 
will follow manufacturer protocols, with daily calibration and quality checks. Certified 
radiographers will conduct all scans. Procedures comply with IRMER (British Institute of 
Radiology, 2015) and University safety guidelines, with radiation exposure kept as low as 
reasonably practicable (ALARP) and considered negligible.  
The following data will be measured: 
− Resting Metabolic Rate (Kcal/day)  
− Relative Skeletal Muscle Index (kg/m2)  
− Total Body Water (L)  
− Intracellular Water (L)  
− Extracellular Water (L)   

 
 
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− 
Total Fat Mass (kg) 
− 
Total Lean Mass (kg) 
− 
Percentage body fat (%)    
Percentage body fat will be calculated using the following formula:   
Percentage Body Fat= (
𝑇𝑜𝑡𝑎𝑙 𝑓𝑎𝑡 𝑚𝑎𝑠𝑠 (𝑘𝑔)
𝑇𝑜𝑡𝑎𝑙 𝑏𝑜𝑑𝑦 𝑚𝑎𝑠𝑠 (𝑘𝑔)) × 100 
Where:   
Total Body Mass (kg) is the sum of Total Fat Mass (kg) and Total Lean Mass (kg).   
 
8.3.4 Peripheral and central vascular function 
The following vascular measures will be assessed at baseline to characterize cerebrovascular 
and peripheral endothelial function: 
− Cerebrovascular reactivity (CVR) of the middle cerebral artery 
− Flow-mediated dilation (FMD) of the brachial artery 
 
8.3.4.1 Cerebrovascular Reactivity (CVR) 
CVR will be assessed using transcranial Doppler ultrasonography (Multidop T, DWL 
Compumedics Germany GmbH) to measure cerebral blood flow (CBF) velocity in the middle 
cerebral artery (MCA; Seigworth et al., 2025). Participants will be positioned either supine or 
seated in a quiet, temperature-controlled environment. A 2-MHz probe will be placed at the 
temporal window to insonate the MCA. Both left and right MCAs will be insonated, and the side 
of head with the highest quality spectral waveform will be selected for analysis.  
Baseline MCA velocity will be recorded for 2–3 minutes under resting conditions. CVR will be 
elicited using a standardized breath-hold protocol. Participants will first breathe normally through 
the nose for one minute to establish normocapnia, after which they will pinch their nose and 
perform a 30-second breath hold. At the end of the maneuver, participants will be instructed to 
exhale through the nose to allow capture of peak end-tidal CO₂ (EtCO₂), before resuming 
normal breathing. Continuous recording of CBF velocity and EtCO₂ will be obtained throughout. 
Baseline MCA velocity will be defined as the average during the normocapnic period, and peak 
velocity will be defined as the average of the highest values at peak EtCO₂. CVR will be 
calculated as the percent change in MCA velocity from baseline to peak divided by the absolute 
change in EtCO₂, expressed as %/mmHg (Liu et al., 2013).  
 
8.3.4.2 Flow-Mediated Dilation (FMD) 
Endothelial function will be evaluated via ultrasound-based measurement (Vivid IQ, GE 
Healthcare, Chalfont St Giles, UK) of brachial artery FMD, following established guidelines 
(Corretti et al., 2002). Participants will rest supine in a quiet, temperature-controlled room for at 
least 10 minutes prior to imaging. 
Baseline brachial artery diameter and flow velocity will be recorded above the antecubital fossa. 
A pneumatic cuff placed on the forearm will be inflated to at least 50 mmHg above systolic blood 
pressure (minimum 200 mmHg) for 5 minutes to induce arterial occlusion. Upon rapid cuff 
release, artery diameter will be recorded continuously for 2–3 minutes to determine the peak 
post-occlusion response. FMD will be calculated as the percentage change from baseline to 
peak diameter (Thijssen et al., 2008). 
 
8.4 Clinical sample collection procedures 

 
 
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8.4.1 Venous blood collection 
The researcher will clean the venipuncture site thoroughly using an alcohol-based antiseptic 
and allow it to dry completely to prevent contamination. A tourniquet will be applied to aid vein 
visualization. Once the vein is located, a sterile needle will be inserted using standard 
phlebotomy technique at a shallow angle (approximately 15–30 degrees) to ensure smooth 
entry and minimize discomfort. 
One 2 mL blood sample will be drawn into a purple topped, Ethylenediaminetetraacetic acid 
(EDTA)-coated tube (Uk Medi). The tube will be labeled with the participant's unique 
identification number and time of collection. 
 
8.4.2 Stool sample collection 
Participants will be provided with FTA faecal sample collection cards (Botnen et al., 2023) at the 
screening visit (week –1) and again at visit 3 (week 11). Each kit will include the FTA card, 
disposable gloves, and clear written instructions for proper at-home sample collection. 
Participants will collect the stool sample by applying a small amount to the card, allow it to dry 
thoroughly, and return it to the study site in the pre-supplied protective envelope. Due to the 
preservative properties of the FTA card, refrigeration or ice packs are not required during 
transport. 
8.5 Storage and analysis of clinical samples 
It is the responsibility of the trial site to ensure that samples are appropriately labelled in 
accordance with the trial procedures to comply with the 1998 Data Protection Act. Biological 
samples collected from participants as part of this trial will be transported, stored, accessed and 
processed in accordance with national legislation relating to the use and storage of human 
tissue for research purposes and such activities shall at least meet the requirements as set out 
in the 2004 HTA. 
 
Table 4: Biological Sample Collection Details 
Purpose 
Visit 1 
(Week –1) 
Visit 2 
(Week 0) 
Midpoint 
(Week 6) 
Visit 3 
(Week 11) 
Visit 4 
(Week 
12) 
Venous Blood Samples 
Cytokine Concentration 
Platelet Activity 
2 x 2mL 
 
2 x 2mL 
2 x 2mL 
 
Fingertip Capillary Blood Samples 
Oral Glucose Tolerance 
Testing 
3 x 1 µL 
 
 
3 x 1 µL 
 
Lipid Assessment 
1 x 40 µL 
 
1 x 40 µL 
1 x 40 µL 
 
HbA1c Assessment 
1 x 1.5 µL 
 
1 x 1.5 µL 
1 x 1.5 µL 
 
Fasting Blood Glucose  
1 x 1 µL 
 
1 x 1 µL 
1 x 1 µL 
 
Stool Samples 

 
 
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Gut Microbial Analyses 
 
1 x5g 
 
1x5g 
 
 
8.5.1 Venous Blood Samples 
Venous blood samples will be collected at Visit 1 (Week –1), Visit 3 (Week 6) and Visit 4 (Week 
11) for the assessment of inflammatory cytokine concentration and markers of platelet activity. 
Each participant will provide one venous blood sample per visit (total: 3 samples per 
participant). 
 
8.5.1.1 Processing and storage  
Samples will be processed within 2 hours of collection: centrifuged at 1,500 × g for 10 minutes 
at 4°C. Plasma aliquots will then be immediately transferred to pre-labeled cryotubes and stored 
at −80°C until batch analysis to minimize freeze-thaw cycles. All pre-analytical handling will 
follow the LBU CSS Biospecimen Handling SOP. 
 
8.5.1.2 Analysis 
Cytokine concentrations will be from plasma derived from venous blood samples, using high-
sensitivity enzyme-linked immunosorbent assay (ELISA) (Thermo Fisher Scientific). The 
following pro-inflammatory and anti-inflammatory cytokines will be quantified:  
− 
IL-6 
− 
TNF-α 
Markers of platelet activity will also be assessed from venous blood samples using validated 
and appropriate laboratory methods. 
 
All assays will be performed using validated high-sensitivity ELISA kits, where applicable, with 
known lower limits of detection and quantification appropriate for low circulating levels of 
biomarkers in plasma. Each sample will be run in duplicate, and mean concentrations will be 
used for analysis. Absorbance will be read using a Multiskan™ GO Microplate 
Spectrophotometer (Thermo Fisher Scientific) at the wavelength specified by the kit protocol. 
Raw absorbance data will be analyzed using SkanIt™ Software, applying a 4-parameter logistic 
(4PL) regression model to generate standard curves and interpolate sample concentrations. 
Standard curves must achieve R² ≥ 0.99 for data to be considered valid. Internal controls and 
reference standards provided with the kits will be included on each plate to ensure inter-assay 
comparability. Inter-assay and intra-assay coefficients of variation (CVs) will be calculated. CVs 
exceeding 15% will trigger a review and potential re-analysis of affected samples. 
 
8.5.1.3 Documentation and Destruction 
Sample tracking will include collection time, storage events, and analysis outcomes. Samples 
will be retained for up to 1 year's post-analysis, unless otherwise stated in the ICF. Samples will 
then be destroyed securely under institutional biosafety policy. No shipping is planned unless 
deemed necessary, in which case samples will be sent on dry ice with appropriate 
documentation. 
 
8.5.2 Capillary Blood Samples 
Capillary blood samples will be collected at Visit 1 (Week –1), Visit 3 (Week 6) and Visit 4 
(Week 11) for the assessment of glucose metabolism, glycated haemoglobin (HbA1c), and lipid 
profile. Each participant will provide seven capillary blood samples per visit (total: 14 samples 

 
 
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per participant). 
 
8.5.2.2 Processing and storage  
All capillary samples will be analysed immediately at the point of care using integrated analyser 
systems. No biological material will be stored, and samples will be disposed of in accordance 
with institutional biosafety and clinical waste protocols immediately after analysis. No samples 
will be retained, transported, or biobanked. 
 
8.5.2.3 Analysis 
All capillary blood analyses will be performed using CE-marked point-of-care diagnostic devices 
to ensure standardisation and accuracy. These methods are outlined in section 8.3.2.  
 
8.5.3 Stool Samples 
Stool samples will be collected at Week 0 and Week 12 for gut microbiome analysis. 
8.5.3.2 Processing and storage  
Upon return to the study site, samples will be stored at room temperature. Samples will be 
temporarily stored under these conditions for up to 1 week, after which they will be shipped to 
Cultech Ltd. (Port Talbot, UK) for microbiome analysis. All shipments will be tracked and include 
full chain-of-custody documentation.  
 
8.5.3.2.1 Shotgun metagenomic sequencing  
Genomic DNA will be recovered from faecal samples preserved on FTA cards. Sections of the 
cards containing stool will be excised, and DNA will be extracted using an appropriate protocol 
in accordance with the manufacturer’s instructions. DNA concentration and purity will be 
assessed fluorometrically, and minimum thresholds for DNA yield and fragment quality will be 
applied prior to sequencing. 
 
Shotgun metagenomic sequencing will be conducted on a high-throughput Illumina platform, 
generating paired-end reads. Raw reads will undergo quality control, including trimming of 
adapters and low-quality bases, and filtering of short reads. Host-derived sequences will be 
removed by alignment to the human genome reference (GRCh38; Langmead & Salzberg, 
2012), and only non-host reads will be retained for downstream analysis. 
 
8.5.3.3 Analysis 
 
8.5.3.4 Taxonomic profiling  
Taxonomic classification of microbial reads (bacteria, archaea, viruses, fungi) will be performed 
using a k-mer–based approach (e.g., Kraken2 or equivalent; Wood et al., 2019), using a curated 
reference database. Species-level abundance estimates will be refined using an appropriate 
algorithm (e.g., Bracken; Lu et al., 2017), with filtering thresholds applied to exclude rare taxa 
from downstream analysis. 
Presence of probiotic strains included in the investigational product will be evaluated using 
strain-resolved genomic methods, comparing mapped reads to available reference genomes. A 
predefined coverage threshold will be used to determine strain-level detection. 
Specific analytical tools and pipelines may be updated at the time of analysis to reflect current 
best practice and advancements in metagenomic analysis.  

 
 
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8.5.3.4 Documentation and Destruction 
 Each sample will be assigned a unique identifier and logged in the study’s sample tracking 
system. Any residual stool material will be destroyed by Cultech according to their biosafety 
disposal protocols following analysis, unless otherwise specified in the ICF. Participants will be 
asked to provide informed consent for the use and transfer of stool samples for microbiome 
research. 
 
 
9. TRIAL TREATMENTS 
 
9.1 Name and description of product under investigation 
Test product name: Lab4P Probiotic Consortium, Cultech LTD.  
The investigational product used in this study is the Lab4P Probiotic Consortium, a multi-strain 
live microbial product supplied in capsule form. The test product contains per serving:  
A total of 5 × 10¹⁰ colony forming units (CFU), comprising the following five bacterial strains:   
− 
Lactobacillus acidophilus CUL60 (NCIMB 30157)   
− 
Lactobacillus acidophilus CUL21 (NCIMB 30156)   
− 
Lactobacillus plantarum CUL66 (NCIMB 30280)   
− 
Bifidobacterium bifidum CUL20 (NCIMB 30153)  
− 
Bifidobacterium animalis subsp. lactis CUL34 (NCIMB 30172)  
 
In addition to probiotic strains, each capsule also contains the following micronutrients at 100–
200% of the recommended nutrient reference value (NRV): 
− Vitamin D – 10 µg (200% NRV) 
− Vitamin C – 80 mg (100% NRV) 
− Zinc – 10 mg (100% NRV) 
 
Lab4P is a patented and marketed product it has been approved for use in humans. It is 
categorised as a food supplement and is available to purchase in the marketplace without 
prescription. Capsules are produced by Cultech LTD, Baglan Industrial Park, Unit 34 Aberavon 
Rd, Port Talbot SA12 7DJ. Both the investigational product and the placebo will be packaged in 
an identical manner. 
 
9.2 Storage and supply 
 
9.2.1 Storage and shipment to sites  
The investigational product and matching placebo will be shipped under controlled conditions to 
the trial site. Upon delivery, research personnel must verify the contents of the shipment against 
the packing list and document receipt using trial-specific accountability forms. These forms will 
serve as source documents and must be completed in real time by the investigator or a 
delegated and trained staff member. While controlled conditions are not required for short-
distance transport, any visible damage to the packaging or concerns regarding product integrity 
must be recorded and reported to the supervisory team immediately. 

 
 
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9.2.2 Accountability  
The research team must maintain accurate records of the investigational product and placebo 
received from Cultech. These records will include receipt and dispensing dates, and dates of 
return and/or destruction of unused supplies. During the course of the trial and at the 
termination, clinical supplies must be accounted for and a written clarification provided in case 
of discrepancies. All dispensing, use, returns, and other dispositions of the investigational and 
placebo products must be recorded in the accountability log. Trial monitors will review 
accountability records during routine monitoring visits to ensure compliance with protocol 
requirements. 
 
The investigational and placebo products must only be used as specified in the trial protocol. 
Under no circumstances may site staff distribute the trial medication to external investigators, 
healthcare services, or use it for purposes outside of this study. 
 
9.2.3 Storage at trial site 
All trial products must be stored under refrigeration at 4–8°C to maintain product stability, as 
indicated by the manufacturer. All products must be stored in a dedicated, secure, temperature-
monitored refrigerator with restricted access. Storage conditions, including daily temperature 
logs, must be recorded and maintained in the investigator site file. 
 
9.2.4 Transportation and storage at subject home  
At the baseline visit, participants will receive both verbal and written instructions on proper 
storage and administration of the investigational product. Capsules must be stored in a 
household refrigerator at a temperature between 4–8°C. Participants will be advised not to 
freeze the capsules and to avoid storing them in warm or humid environments. If refrigeration is 
temporarily unavailable (e.g., during travel), participants will be instructed to store the capsules 
in a cool, dry location and return them to refrigeration as soon as possible.  
Study staff will reinforce these instructions at all contact points and will discuss any barriers to 
proper storage, daily adherence, or timing of administration. All deviations from recommended 
storage or administration will be documented and reviewed by the research team.  
 
9.2.5 Return and destruction of product 
Returned medication will be checked for compliance and documented accordingly. Any 
remaining investigational product or placebo will be collected by the PI, repackaged in a tamper-
evident container, and stored securely at the trial center until the conclusion of the study. All 
unused supplies will be properly destroyed at the trial site or sent back when indicated by 
Cultech. Written authorization must be obtained from Cultech prior to the destruction which must 
be documented. 
 
9.3 Preparation and labelling of Investigational Product 
Both the investigational product and the placebo are packaged in induction-sealed, opaque 
plastic pots to prevent exposure to light and moisture and to maintain product stability 
throughout the study period.   
Minimum information to be included on the trial product label will be:  
− 
Study Title: ProCOG 

 
 
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− 
Participant Number: To be entered at the time of dispensing 
− 
Contents: 90 capsules 
− 
Investigator: Lewis Hepburn, Principal Investigator 
− 
Investigator Contact: L.Hepburn3449@student.leedsbeckett.ac.uk 
− 
The statements: “For study purposes only.” “Keep out of reach of children.” 
− 
Expiry date: To be added according to the product lot supplied 
− 
Batch number: To be added according to the product lot supplied 
− 
Manufacturer: Cultech Ltd, United Kingdom. 
− 
Statements pertaining to dosage, administration and storage information, 
including: “Take one capsule per day with food (with or without a drink).” “Avoid hot 
drinks at the time of ingestion.” “Take with any meal during the day.” And “Store 
capsules in a refrigerator.”  
 
9.4 Dosage schedules 
Participants will consume one capsule per day for 3 months (90 days). Participants will be 
instructed to take one capsule, orally once daily, with food, at a consistent time of day. No 
specific preparation is required prior to administration. 
 
The selected dose, route, and duration of administration for the Lab4P Probiotic Consortium are 
supported by existing clinical data on its safety, tolerability, and product stability (Michael et al., 
2020; Michael et al., 2021). Oral administration was selected as it is the most established and 
effective route for probiotic delivery in human studies (Baral et al., 2021).  
 
The 12-week treatment duration was informed by previous research demonstrating the efficacy 
of Lab4P in reducing body weight and improving metabolic parameters within this timeframe 
(Michael et al., 2021). In addition, the 12-week duration is consistent with studies involving other 
probiotic formulations that have reported measurable changes in metabolic and cognitive 
outcomes over similar periods (Mo et al., 2024; Salles et al., 2020; Zhang et al., 2020). This 
duration is also considered a practical compromise to support participant retention and overall 
study feasibility. 
 
9.4.1 Missed Doses 
If a participant forgets to take a dose, they will be advised to take it as soon as they remember. 
If they do not remember until the following day, the missed dose should be skipped and they 
should continue with their usual schedule. Participants should not take two doses on the same 
day to make up for a missed dose. 
 
9.4.2 Vomiting Post-Dose 
If vomiting occurs within 30 minutes of dosing, participants may take a replacement capsule. 
Vomiting after 30 minutes will not require redosing, as sufficient absorption of probiotic 
organisms is likely to have occurred. 
 
9.4.3 Treatment Resumption After Temporary Suspension 
In the event of temporary discontinuation (e.g. due to illness or travel), participants may resume 
daily dosing without dosage adjustment once able to comply with the protocol. Missed days will 
be recorded, but no dose compensation will be applied. 

 
 
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9.5 Known reactions and interaction with other therapies 
The use of medications or treatments that may interfere with the IP, confound key efficacy 
endpoints, or pose safety concerns will be carefully monitored during the study. While 
participants will not be prohibited from using medically necessary treatments, they will be 
instructed to avoid consciously starting new probiotics, prebiotics, synbiotics, or related 
supplements during the study period. All concomitant medications and supplements will be 
recorded and considered during data analysis. 
 
To minimize the risk of interference, individuals with recent or current use of specific agents 
known to impact the gut microbiota, glucose metabolism, or probiotic viability will be excluded at 
screening. Specifically, participants will be excluded if they have used any of the following within 
4 weeks prior to baseline: 
− Antibiotics (oral, intravenous, or topical with potential for systemic absorption), due to 
their impact on gut microbiota composition and potential to reduce probiotic efficacy 
(Éliás et al., 2023). 
− Proton pump inhibitors (PPIs), H2-receptor antagonists, and other agents that alter 
gastric pH, as they may impair probiotic survival (Garg & Mohajeri, 2024; Vila et al., 
2020). 
− Immunosuppressive agents, which may affect probiotic-host interactions (Gibson et 
al., 2020). 
− Systemic corticosteroids or medications known to significantly alter glucose 
metabolism, such as insulin, metformin, sulfonylureas, GLP-1 receptor agonists, β-
blockers, and thiazide diuretics, due to their influence on OGTT outcomes and metabolic 
endpoints (Eyth et al., 2023). 
 
Participants will be advised to report any new medications or supplements taken during the trial. 
Use of agents listed above during the study will be reviewed on a case-by-case basis by the 
research team, recorded in the eCRF and flagged for consideration during analysis. These 
events may be recorded as protocol deviations only if they substantially affect outcome 
interpretation, in line with the monitoring and statistical analysis plans. 
 
9.6 Concomitant medication 
Study subjects may receive any needed medically prescribed treatment/therapy/medication.  
Information regarding medications, treatments or therapies administered, including the name of 
the active ingredient and duration of treatment, will be collected throughout the trial. The start 
date, stop date and indication for concomitant treatments/medications received from ICF 
signature (screening visit) until the last visit (including follow-up).  
 
All concomitant medications, prescription, over-the-counter, herbal, and nutritional supplements, 
must be documented in the relevent eCRF, including the drug name, indication, route of 
administration, dose, frequency, start and stop dates.  
 
9.7 Trial restrictions 
To minimize confounding factors and preserve data integrity, the following pre-visit requirements 
must be followed prior to each in-person assessment: 

 
 
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Table 5: Pre-laboratory assessment requirements 
Requirement 
Details  
Timeframe Before 
visit 
Alcohol restriction 
No consumption of alcoholic beverages 
24 hours 
Fasting and beverage 
restriction 
No food or caloric beverages. Water is 
permitted. 
10 hours 
Vigorous physical activity 
restriction 
No vigorous exercise (e.g., running, high-
intensity cycling, competitive sports). 
10 hours 
Restrictions will be clearly communicated to participants through the PIS, reinforced via the pre-
screening questionnaire, and compliance will be checked verbally at each in-person visit. 
 
In addition, to reduce potential confounding effects on gut microbiota composition, participants 
will be asked to refrain from starting any new probiotic supplements or regularly consuming 
probiotic-rich foods (e.g., yogurt with live cultures, kefir, kombucha, kimchi, sauerkraut, miso, or 
other fermented products containing live bacteria) throughout the study period (from recruitment 
to Week 12 final visit). Participants who already consume these products regularly will be 
eligible to take part but will be asked to discontinue them if they choose to participate, so that 
the study intervention is the sole source of probiotic exposure. All dietary patterns, including 
probiotic food intake, will be recorded at baseline and monitored throughout the study to allow 
for consideration during data analysis. 
 
9.8 Assessment of compliance with treatment  
The research team will record the number of capsules dispensed; the number returned and 
calculate adherence as the proportion of capsules consumed relative to the number expected 
for the treatment period. These data will be entered into the eCRF.  
Product compliance will be assessed at Visit 4 (week 12). To be considered compliant, 
participants must have consumed a minimum of 80% of the assigned study product. 
Participants not meeting this threshold will be included in the ITT population for primary analysis 
and excluded from the PP population unless otherwise justified. Compliance data may also be 
used in secondary or sensitivity analyses, as defined in the Statistical Analysis Plan. 
 
 
10 PHARMACOVIGILANCE 
 
10.1 Definitions 
Term 
Definition 
Adverse Event (AE) 
Any untoward medical occurrence in a participant to whom a 
medicinal product has been administered, including occurrences 
which are not necessarily caused by or related to that product. 
Adverse Reaction 
(AR) 
 
An untoward and unintended response in a participant to an 
investigational medicinal product which is related to any dose 
administered to that participant. 
The phrase "response to an investigational medicinal product" means 
that a causal relationship between a trial medication and an AE is at 
least a reasonable possibility, i.e. the relationship cannot be ruled out. 

 
 
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All cases judged by either the reporting medically qualified 
professional or the Sponsor as having a reasonable suspected causal 
relationship to the trial medication qualify as adverse reactions. It is 
important to note that this is entirely separate to the known side 
effects listed in the SmPC. It is specifically a temporal relationship 
between taking the drug, the half-life, and the time of the event or any 
valid alternative etiology that would explain the event. 
Serious Adverse 
Event (SAE) 
A serious adverse event is any untoward medical occurrence that: 
• results in death 
• is life-threatening 
• requires inpatient hospitalisation or prolongation of existing 
hospitalisation 
• results in persistent or significant disability/incapacity 
• consists of a congenital anomaly or birth defect 
Other ‘important medical events’ may also be considered serious if 
they jeopardise the participant or require an intervention to prevent 
one of the above consequences. 
NOTE: The term "life-threatening" in the definition of "serious" refers 
to an event in which the participant was at risk of death at the time of 
the event; it does not refer to an event which hypothetically might 
have caused death if it were more severe. 
Serious Adverse 
Reaction (SAR) 
An adverse event that is both serious and, in the opinion of the 
reporting Investigator, believed with reasonable probability to be due 
to one of the trial treatments, based on the information provided. 
Suspected 
Unexpected Serious 
Adverse Reaction 
(SUSAR) 
A serious adverse reaction, the nature and severity of which is not 
consistent with the information about the medicinal product in 
question set out in the reference safety information: 
• in the case of a product with a marketing authorisation, this could be in 
the summary of product characteristics (SmPC) for that product, so 
long as it is being used within it’s licence. If it is being used off label an 
assessment of the SmPCs suitability will need to be undertaken. 
• in the case of any other investigational medicinal product, in the 
investigator’s brochure (IB) relating to the trial in question 
 
10.2 Operational definitions for (S)AEs  
 
10.2.1 Identification of (S)AEs 
Adverse events will be identified through: 
− Participant self-report 
− Investigator questioning during study visits 
− Physical examinations and laboratory assessments 
− Other clinically relevant observations 
 
10.2.2 Evaluation of AEs and SAEs 
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and expectedness in accordance with ICH-GCP, the UK Clinical Trials Regulations, and the 
requirements of the approving REC. These will be assessed using the criteria outlined below. 
 
10.2.2.1 Severity 
Category  
Definition 
Mild 
Symptoms hardly perceived, only slight impairment of general well-being. 
Moderate 
Clearly noticeable symptom, but tolerable without immediate relief. 
Severe 
Overwhelming discomfort. 
 
10.2.2.2 Causality 
Investigators will make causality assessments as though the participant received the active 
investigational product, to preserve blinding integrity. 
Category 
Definition 
Unrelated 
An adverse event, which is clearly not related to the product(s) and/or study 
procedure(s). 
Unlikely 
The event is unlikely related to the product(s) and/or procedures if it occurs within 
a temporal relationship to product(s) administration and/or study procedure(s) 
that makes a causal relationship improbable and in which drugs, chemicals or 
underlying disease provide more plausible explanations. 
Probable 
The event is probably related to the product(s) and/or procedures if it occurs in a 
reasonable time sequence to administration of the product(s) and/or study 
procedure(s), and if the event is unlikely to be attributed to concurrent disease, 
drugs or chemicals. The event should follow a clinically plausible response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this 
definition. 
Related 
The event is related to the product(s) and/or study procedure(s) if it occurs in a 
plausible time relationship to product(s) administration and/or study procedure(s), 
and if it cannot be explained by concurrent disease or other drugs or chemicals. 
The response to withdrawal of the product(s) and/or study procedure(s) 
(dechallenge) should be clinically plausible. The event must be plausible 
pharmacologically or phenomenologically. Confirmation must include a 
satisfactory rechallenge procedure. 
 
10.2.2.3 Expectedness 
Category  
Definition 
Expected 
An adverse event which is consistent with the information about the intervention 
clearly defined in this protocol. 
unexpected 
An adverse event which is not consistent with the information about the intervention 
clearly defined in this protocol. 
 
10.2.3 Events to Be Reported to the Sponsor 

 
 
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The following events will be reported to the Sponsor: 
− All SAEs, regardless of causality, within 24 hours of the site becoming aware 
− All SARs, including any unexpected worsening of known adverse reactions 
− Unexpected ARs not consistent with the known safety profile of the investigational 
product 
These events will be reported using the Serious Adverse Event Case Report Form (SAE CRF) 
provided in the Investigator Site File and submitted via secure electronic or fax transmission to 
the Sponsor or designee. 
 
10.2.4 Events Not Requiring Reporting to the Sponsor 
The following will be recorded but not routinely reported to the Sponsor, unless considered 
clinically significant or unexpected in severity, frequency, or duration: 
− Mild gastrointestinal symptoms such as bloating or flatulence 
− 
Expected minor adverse reactions consistent with the known safety profile of probiotic 
supplements 
− Elective or pre-planned hospital admissions not associated with clinical deterioration 
− Admissions for routine treatment or monitoring of the studied indication 
− Outpatient treatment not fulfilling criteria for seriousness 
All non-serious AEs will be recorded in the eCRF. These records will be available to the 
Sponsor and regulatory authorities upon request. 
 
10.3 Recording and reporting of SAEs, SARs AND SUSARs 
AEs and SAEs will be recorded from the time of written informed consent until the final study 
visit at Week 12. ARs, SARs, SUSARs will be recorded and reported from the first 
administration of the investigational product until the final dose is taken, with all relevent events 
captured at the final visit. Upon becoming aware of any SAE, the investigator must enter the 
event into the eCRF within 24 hours, regardless of whether the event is considered related to 
the investigational product. All SAEs occurring up to and including the final trial visit and within 
30 days following the last dose of study product must be reported in this way. Initial reports 
should be submitted even if causality and expectedness cannot yet be determined.  
 
Upon entry of an SAE in the eCRF, an automatic notification will be sent to the PI, who must 
also be directly notified within 24 hours of the investigator becoming aware of the event. The PI 
will review all SAEs and determine the appropriate course of action in collaboration with the 
research supervisory team. The supervisory team, acting on behalf of the Sponsor, will be 
responsible for notifying the LREC and any other relevant regulatory bodies, in line with 
applicable reporting timelines and procedures 
 
Each SAE must be electronically signed by the reporting investigator within the eCRF. 
Expedited reporting requirements apply to SUSARs. Any SUSAR that is fatal or life-threatening 
must be reported to the LREC and MHRA (if applicable) within 7 calendar days of the PI or 
project supervisor becoming aware of the event, with any follow-up information submitted within 
an additional 8 days. All other SUSARs must be reported to the LREC and MHRA within 15 
calendar days of awareness. 
 

 
 
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For each SAE and/or SUSAR, the following information will be collected: 
− 
Subject and date 
− 
Description of event 
− 
Reporting source 
− 
Suspect product 
− 
Duration 
− 
Frequency 
− 
Intensity 
− 
Seriousness 
− 
Action taken 
− 
Outcome and sequelae if any 
− 
Relationship to test product 
If a participant withdraws consent for further data collection, this does not remove the 
requirement to report SARs or SUSARs that occur or become known to the investigator 
following withdrawal, as these are reportable for regulatory and safety purposes. This will be 
clearly explained to participants in the Participant Information Sheet (PIS). 
 
10.4 Responsibilities 
 
PI:  
− Checking for AEs and ARs when participants attend for treatment / follow-up. 
− 
Using medical judgement in assigning seriousness, causality and whether the 
event/reaction was anticipated.  
− Ensuring that all SAEs are recorded and reported to the project supervisor within 
24 hours of becoming aware of the event and provide further follow-up information 
as soon as available.  
− Ensuring that SAEs are chased with the project supervisor if a record of receipt is 
not received within 2 working days of initial reporting.  
− Ensuring that AEs and ARs are recorded and reported to the project supervisor in 
line with the requirements of the protocol.  
 
Project Supervisor: 
− Clinical oversight of the safety of patients participating in the trial, including an 
ongoing review of the risk / benefit. 
− Using medical judgement in assigning the SAEs seriousness, causality and 
whether the event was anticipated (in line with the Reference Safety Information) 
where it has not been possible to obtain local medical assessment. 
− Immediate review of all SUSARs.  
− Expedited reporting of SUSARs to the LREC or Competent Authority (MHRA in 
UK) as necessary and within required timelines. 
− The unblinding of a participant for the purpose of expedited SUSAR reporting. 
 
10.5 Notification of deaths 
In the event that a participant dies during the trial period (from informed consent until the final 
study visit at Week 12), the project supervisor will assess whether the death is related to study 

 
 
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participation. Only deaths deemed related to the investigational product will be reported to the 
LREC, and in no case later than 24 hours from awareness. 
Deaths unrelated to the IMP are not routinely reportable but will be documented in the 
participant’s medical records, trial documentation, and reported in the final study report, where 
applicable. Any unexpected deaths, regardless of relatedness, will be flagged for review by the 
project supervisor and reported to the LREC if deemed relevant to participant safety. 
 
10.6 Pregnancy reporting 
Although pregnancy should not be considered as an AE/SAE, any pregnancies will be captured 
using the participants CRF and followed up as if they were SAEs to determine the outcome, 
especially in the case of the pregnancy going to term to determine the outcome with respect to 
the baby. 
 
10.7 Reporting urgent safety measures 
If any urgent safety measures are taken the PI shall immediately and, in any event, no later than 
3 days from the date the measures are taken, give written notice to the supervisory team and 
the relevant LREC of the measures taken and the circumstances giving rise to those measures. 
 
10.8 The type and duration of the follow-up of participants after adverse 
reactions. 
All SAEs must be followed up until the outcome is resolved or stable. In case of SAE(s) 
persisting beyond trial termination, a follow-up visit may be required. If further analyses are 
required for the evaluation of a potential cause-effect relationship between the investigational 
product and/or procedures and the AE, all examinations and laboratory analyses will be 
documented in the (e)CRF or in an attached file. 
 
11 STATISTICS AND DATA ANALYSIS 
 
11.1 Sample size calculation 
The sample size calculation is based on the study’s primary outcome: performance on a verbal 
memory task. Given the exploratory nature of the trial, the sample size was informed by effect 
sizes reported in comparable studies. Two previous trials investigating probiotic interventions 
versus placebo on verbal memory performance tasks reported moderate effect sizes of 0.60 
and 0.67, respectively (Önning et al., 2023; Schneider et al., 2023). While the study populations 
in those trials were not directly comparable to the population targeted in the present study, they 
provide a reference point for estimating effect size in the absence of directly relevant prior data. 
Based on this, a conservative average effect size of 0.63 was selected for power analysis.   
Using G*Power software (version 3.1.9.7), a sample size calculation was conducted based on 
an independent samples t-test, with equal group allocation, a one-tailed alpha of 0.05, and 80% 
power. The resulting required sample size was 64 participants (32 per group) to detect the 
specified effect size. To account for an estimated attrition rate of 10%, this study will aim to 
enroll 70 participants.   
 
11.2 Planned recruitment rate 
This study aims to recruit a total of 70 participants over two recruitment waves, each lasting 

 
 
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approximately 3 months, for a total recruitment period of 6 months. As no formal feasibility study 
has been conducted, these estimates are based on anticipated levels of interest and response 
to similar recruitment strategies used in health research. 
 
It is anticipated that each recruitment wave will generate 20–30 expressions of interest per 
week. Based on the expected inclusion/exclusion profile, we estimate a screening eligibility rate 
of approximately 50–60%. Of those eligible, a consent rate of 70–80% is expected. This 
corresponds to an expected recruitment rate of 3–5 participants per week, with a maximum of 
10 per week, per wave. 
 
Assuming a consistent response across both waves, we anticipate completing recruitment 
within the 6-month timeline. This staggered approach allows for recruitment monitoring and, if 
needed, protocol refinements or additional outreach strategies between waves. Participant flow, 
eligibility rates, and retention will be reviewed regularly. Adjustments to recruitment materials or 
procedures will be made in consultation with the research team if accrual falls below target. 
 
11.3 Statistical analysis plan 
 
11.3.1 General 
All statistical tests will be two-sided, with a significance level of p < 0.05, unless otherwise 
specified. Where appropriate, results will be accompanied by 95% confidence intervals and 
effect size estimates (e.g., Cohen’s d, partial η²). All statistical analyses will be conducted using 
SPSS (Version 30.0.0). 
 
11.3.2 Summary of baseline data and flow of patients 
Baseline data will be summarised descriptively to assess the comparability of treatment groups 
at randomisation. No formal statistical tests will be conducted on baseline variables. Summary 
statistics will be presented by treatment group, with an additional "Total" column to reflect 
overall sample characteristics. 
 
Data will be summarised according to variable type: means and standard deviations (SD) for 
normally distributed continuous variables, medians and interquartile ranges (IQR) for skewed 
distributions, and frequencies and percentages for categorical variables. Analyses will be 
conducted on the treated set (participants who received at least one dose of the investigational 
product), with additional analysis sets used as appropriate, depending on context. 
 
A CONSORT-compliant participant flow diagram will be provided, detailing the number of 
participants screened, eligible, consented, randomised, treated, completing follow-up, and 
included in the final analysis. 
 
11.3.3 Primary outcome analysis 
The primary analysis will assess the effect of Lab4P probiotic supplementation compared to 
placebo on verbal memory performance, measured by accuracy on the CANTAB VRM task, 
from baseline to Week 12. 
 
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(baseline, Week 6, and Week 12). Fixed effects will include time, treatment group (Lab4P vs. 
placebo), and their interaction (time × group), which represents the primary effect of interest. A 
random intercept will be included for each participant to account for within-subject correlation. 
Baseline VRM scores will be included as a covariate where appropriate. 
 
Model assumptions will be assessed and addressed using standard diagnostic procedures. 
Results will be reported as estimated mean differences in change between groups over time, 
with 95% confidence intervals and two-sided p-values, using a significance threshold of p < 
0.05. 
 
No adjustments for multiple comparisons are required, as verbal memory performance is the 
sole primary outcome. 
 
11.3.4 Secondary outcome analysis 
Secondary outcome analyses will explore the effect of Lab4P probiotic supplementation 
compared to placebo on a range of cognitive, sleep, cardiometabolic, vascular, inflammatory, 
anthropometric, body composition (outlined in section 3.4). These analyses are considered 
exploratory and hypothesis-generating, as the trial is not powered to detect statistically 
significant effects in these outcomes. 
 
For secondary outcomes measured at three time points (e.g., cognitive and sleep measures, 
cardiometabolic data, anthropometric data), change over time will be assessed using linear 
mixed-effects models with fixed effects for treatment group, time, and their interaction (time × 
group), and a random intercept for participant to account for repeated measures. Where 
appropriate, baseline values of the outcome variable will be included as covariates. Non-
normally distributed outcomes may be transformed or modelled using alternative techniques 
(e.g., generalized linear models). 
 
For outcomes measured only at baseline and week 12 (e.g., glucose tolerance, cytokines, DXA 
metrics), between-group differences in change from baseline will be assessed using delta 
scores, adjusting for baseline values. Dietary intake data from the food diaries will be 
summarised and analysed using suitable statistical techniques, selected based on the 
characteristics of the data and distributional assumptions. For gastrointestinal and systemic 
symptom occurrence, as measured by the 14-day recall symptom questionnaire, outcomes will 
be treated as count data (i.e., number of days with each symptom out of 14). Analyses will use 
generalized linear models with an appropriate distribution (e.g., negative binomial or Poisson, 
depending on dispersion) to compare symptom frequency between groups at week 12, 
adjusting for baseline symptom frequency. 
 
Estimates of between-group differences in change over time will be reported as mean 
differences with 95% confidence intervals, along with associated two-sided p-values. However, 
formal hypothesis testing will not be emphasised, and no adjustment for multiple comparisons 
will be applied given the exploratory nature of these analyses. 
 
11.3.5 Microbial Diversity analysis 
 
11.3.5.1 Alpha Diversity 

 
 
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Microbial diversity will be assessed to explore differences in gut microbiota composition 
between the probiotic and placebo groups. Both alpha diversity (within-sample diversity) and 
beta diversity (between-sample diversity) metrics will be calculated. 
 
Analyses will focus on species-level profiles and may include standard ecological measures 
such as Shannon diversity, Bray–Curtis dissimilarity, and ordination techniques (e.g., NMDS). 
Statistical comparisons between groups will account for relevant covariates including age, BMI, 
and menstruation status. Multivariate models may be used to examine group-level differences in 
community structure and dispersion, incorporating appropriate correction for multiple 
comparisons. 
 
Differential abundance testing will be conducted where appropriate to identify microbial taxa 
with significantly different relative abundance between groups. 
 
11.4 Subgroup analyses  
Subgroup analyses based on sex and ethnicity will be explored by incorporating group × time × 
subgroup interaction terms within mixed-effects models or through stratified analyses, as 
appropriate. These exploratory analyses are hypothesis-generating in nature and will be 
interpreted with caution given the limited statistical power and the absence of adjustments for 
multiple comparisons. 
 
11.5 Adjusted analysis 
Adjusted analyses are not planned as part of the primary analysis, which will rely on the 
assumption that randomisation ensures baseline comparability. However, adjusted models may 
be included as sensitivity analyses if substantial baseline imbalance is observed or to improve 
precision. Potential covariates for adjustment include age, sex, BMI, and baseline scores for the 
outcome variable. All adjustments will be pre-specified in the relevent section. The primary 
analysis will remain unadjusted. 
 
11.6 Interim analysis and criteria for the premature termination of the trial 
No formal interim analysis is planned for this study. However, interim review of recruitment, 
retention, adherence, and safety data may be conducted by the PI in collaboration with the 
academic supervisors, where appropriate. These reviews will focus on operational aspects of 
the study (e.g. participant flow, data completeness, protocol adherence) and will not involve 
formal statistical comparisons between treatment groups. All reviews will be conducted using 
blinded unless unblinding is specifically justified and approved by the project supervisor. 
There are no predefined statistical stopping rules for efficacy, harm, or futility. However, the PI, 
in consultation with the project supervisor may recommend pausing or terminating the trial early 
in the event of: 
− Emerging safety concerns or unexpected adverse events 
− Significant protocol violations or operational challenges compromising trial integrity 
− Poor recruitment or retention making it unlikely the trial can meet its objectives 
Any such decisions will be made on the basis of judgment, documented review of available 
data, and oversight from the supervisory team. In the absence of a formal DMC, ethical 
oversight will be maintained through ongoing supervisory review and required reporting to the 
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11.7 Participant Population 
 
11.7.1 Full analysis dataset 
The full analysis dataset (FAS) will include all the randomized subjects, except those that never 
started the trial, or subjects who failed to satisfy study entry eligibility criteria or had no post-
randomization data. The FAS will be analyzed according to the assigned group 
per randomization. The Full Analysis Set (FAS) will be used for primary and secondary 
analyses, following the intention-to-treat (ITT) principle. 
 
11.7.2 Per-Protocol analysis dataset 
The Per-protocol (PP) analysis dataset will consist of subjects included in the FAS, without any 
departures from the protocol which are believed to impact the analyses of interest. The subjects 
with major protocol deviations related to GCP (e.g. ICF process not followed) will not be 
removed from PP by default. A separate listing of these subjects will be provided in the 
statistical report. A list of protocol violations that would exclude subjects from inclusion in the PP 
analysis dataset will be specified during the blind data review meeting. A Per-Protocol (PP) 
analysis will be conducted as a sensitivity analysis, including only participants who complete the 
study without major protocol deviations. 
 
11.7.3 Safety analysis set 
Safety population includes all randomized subjects that started the trial, classified according to 
the product received. The Safety Set will be used for safety and tolerability assessments. 
 
11.8 Procedure(s) to account for missing or spurious data 
Missing values for the primary endpoint may be imputed or completely handled using maximum 
likelihood approaches. Unless another pattern of missingness is demonstrated during the review 
of the data, missing data will be assumed to be missing at random. Handling of missing values 
will be refined and finalized during the blind review of the data. Exploration of outliers will be 
carried out at the blind review of the data. If outliers are detected on any of the endpoints, robust 
approaches or sensitivity analysis would be used.  
 
11.9 Other statistical considerations. 
A detailed SAP will be developed prior to the final analysis. The SAP will be reviewed and, if 
necessary, revised following a blinded review of the data to address any outstanding analytical 
considerations. The final version of the SAP will be signed off prior to unblinding to ensure the 
integrity of the analysis. 
 
12. DATA MANAGEMENT  
 
12.1 Data collection tools and source document identification 
12.1.1 Case Report Forms 
All study data will be captured using structured Case Report Forms (CRFs), which function as 
the primary tools for recording protocol-specified variables across both on-site and remote visits. 
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and dated by the PI, handwritten or electronic signature as appropriate. Paper CRFs are printed 
in duplicate and are filled out in black ink. If an entry on a paper CRF requires a change, the 
correction must be made so that the corrected item remains legible. When eCRFs are used, 
corrections are recorded in an audit trail and the data are extracted from the eCRF database 
after final check and signature.  
CRFs will be developed in accordance with the visit schedule and outcome measures defined in 
this protocol. The Investigator and/or designee will accurately, completely, and in a timely 
manner record data resulting from the execution of the protocol on paper CRFs or eCRFs, 
respectively). One CRF will be completed for each participant. Data will be entered into CRFs 
either by direct transcription from source documents or imported from validated electronic 
systems where applicable. Each data point collected for this study will have a verifiable source. 
 
12.1.2 Source documents 
For this study, the following data sources are identified:  
 
12.1.2.1 Investigator’s site data 
Site-collected data will include anthropometric measurements, anthropometric measurements, 
point of care cardiometabolic markers, OGTT timepoints and glucose values, and the outcomes 
of FMD and CVR assessments. Such data will be recorded directly into the eCRF by authorised 
site staff at the time of collection. In these cases, the eCRF will serve as the source document 
(see section 12.1.2.5). 
Other site assessments, such as sample collection logs and blood sampling details, may initially 
be recorded on paper or site worksheets and subsequently transcribed into the eCRF. In such 
cases, the original paper documentation will constitute the source document and must be 
retained in the participant file for monitoring and audit purposes. 
 
12.1.2.2 Patient reported outcomes 
Self-completed questionnaires including the PSQI, demographic and lifestyle questionnaires, 
and any symptom or adverse event reports, will be collected electronically using the Qualtrics 
platform. All responses will be linked to pseudonymised participant ID numbers and exported in 
coded format. The Qualtrics export files will serve as the source documents for these data. 
 
12.1.2.3 External data and laboratory data 
Body composition, biochemical and stool samples will be analysed at LBU, Cultech LTD or by 
an accredited third-party laboratory. All results will be transferred securely in coded format and 
merged into the clinical dataset. Laboratory reports will serve as the source documents for these 
data. 
 
12.1.2.4 Cognitive Assessment Data (CANTAB Platform) 
All CANTAB task performance data will be automatically recorded by the software and exported 
in pseudonymised, coded format. These electronic output files will constitute the source data for 
cognitive outcomes and will be imported into the study database for analysis. 
 
12.1.2.5 Case Report Forms as source Documents 
The eCRFs will serve as source documents in specific instances where data are recorded 
directly by study personnel during the visit, including anthropometric measurements (height, 

 
 
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weight, waist and hip circumference), point of care cardiometabolic markers (HbA1c, blood 
pressure, lipid profile) the OGTT timepoints and glucose values, and the outcomes of FMD and 
CVR assessments. In these cases, the eCRF will constitute the first and only recording of the 
data, and thus will be considered the source document. For all other data, the eCRF will not be 
considered a source document, and the original record (e.g., paper forms, electronic platform 
exports, or laboratory reports) will serve as the source and must be retained accordingly. 
 
12.2 Data handling and record keeping 
 
12.2.1 Audit trail and eSignature 
All electronic data entry systems used in this trial will include audit trail functionality to ensure 
full traceability of data entry, edits, and corrections. The audit trail will capture the date, time, 
user ID, and reason for changes made. Where electronic signatures are used (e.g., for consent 
or data verification), they will comply with HRA standards for eSignatures and data integrity. 
 
12.2.2 Data validation 
Data will undergo routine validation checks at the point of entry and during scheduled 
monitoring visits. Validation procedures include range checks, logic checks, and cross-
verification against source documents. Any data queries will be resolved and documented 
through the data query management system prior to database lock. 
 
12.2.3 Coding 
Type of data 
Dataset 
Dictionary 
Adverse Events 
AE 
MedDRA version 24 
Serious Adverse Events 
SAE 
MedDRA version 24 
 
12.2.4 Database Lock 
The database will be locked following completion of data cleaning, resolution of all queries, and 
final study visits. Once locked, no further changes will be made to the dataset. The research 
team will only receive access to unblinded treatment codes after database lock to maintain data 
integrity. 
 
12.3 Access to Data 
Access to study data will be limited to authorised personnel involved in the conduct, monitoring, 
and oversight of the trial. These include the PI, designated research staff, approved monitors, 
and auditors from LBU or relevant regulatory authorities. All individuals accessing the data must 
be trained in data protection and trial-specific confidentiality protocols. Access will be managed 
via user-specific logins with role-based permissions. 
Direct access will be granted to authorised representatives from the Sponsor, host institution 
and the regulatory authorities to permit trial-related monitoring, audits and inspections- in line 
with participant consent. 
 
12.4 Archiving 
Archiving will be authorised by the project supervisor following submission of the End of Trial 

 
 
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Report. The PI will retain responsibility for archiving the trial master file, final protocol, 
participant-facing documents, regulatory correspondence, and the final locked database. The PI 
will be responsible for archiving the Investigator Site File, signed ICFs, source documents, and 
completed CRFs. 
All essential documents, including the trial database and associated metadata, will be retained 
for a minimum of five (5) years after completion of the trial, in accordance with GCP and 
applicable regulations. Records will be stored securely in an access-controlled environment with 
appropriate conditions for document preservation. No essential documents may be destroyed 
without prior written authorisation from the project supervisor. The project supervisor will ensure 
that archiving responsibilities are fulfilled and documented. 
 
 
13. MONITORING, AUDIT & INSPECTION 
Throughout the trial and following its completion, the PI will permit oversight activities by the 
Sponsor or its designated representatives. This may include trial monitoring, audits, and 
regulatory inspections. 
 
Monitoring may be conducted periodically by the project supervisor and/or university-appointed 
personnel to review the progress of the trial, verify the accuracy and completeness of (e)CRFs, 
and ensure compliance with the protocol, GCP guidelines, and applicable regulations. The PI is 
responsible for ensuring (e)CRFs are completed regularly and are up to date before each 
monitoring activity. 
 
Audits may be conducted by internal university representatives or external auditors acting on 
behalf of the Sponsor. The aim of an audit is to evaluate trial conduct, compliance with GCP and 
Sponsor procedures, and the integrity of the trial data. The PI and study team are expected to 
be available during audits, cooperate fully, and provide access to source documents, (e)CRFs, 
and other relevant trial documentation. The PI will be contacted in advance to schedule any 
audit visit. 
 
Regulatory Inspections may be carried out by government authorities, research ethics 
committees, or institutional bodies (e.g., MHRA, LREC). These inspections serve to 
independently assess trial conduct, documentation, and facilities. The PI and relevant staff must 
be available and provide full access to all necessary trial records. In the event of prior notice of 
an inspection, the PI must notify the Sponsor immediately. 
 
14. ETHICAL AND REGULATORY CONSIDERATIONS 
 
14.1 Research Ethics Committee (REC) review & reports 
Prior to the commencement of the trial, ethical approval will be sought from the appropriate 
Local Research Ethics Council (LREC) at LBU. This review will cover the trial protocol, PIS, ICF, 
and all other relevant documents, such as recruitment advertisements. No substantial 
amendments to the protocol or supporting documentation will be implemented until the LREC 
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All correspondence with the LREC will be retained in the Trial Master File (TMF), in accordance 
with LBU’s research governance procedures. The PI will notify the LREC of the end of the trial. 
If the trial is ended prematurely, the PI will provide notification to the REC, including a statement 
outlining the reasons for early termination. Within one year of trial completion, a final report will 
be submitted to the REC, detailing the study results and including any relevant publications or 
abstracts. 
 
14.2 Regulatory Compliance 
This trial will be conducted in compliance with the International Conference on Harmonization 
(ICH) guidelines and the ethical principles that have their origin in the Declaration of Helsinki 
and its subsequent amendments (World Health Organization, 2005; World Medical Association, 
2014). The study will also adhere to the UK Policy Framework for Health and Social Care 
Research (Health Research Authority, 2025), the Data Protection Act 2018, and the UK GDPR. 
Initiation of the trial is contingent upon receipt of all required regulatory approvals, including a 
favourable opinion from the LREC. 
 
As the study involves exposure to ionising radiation through iDXA, it falls under the scope of the 
Ionising Radiation Medical Exposure Regulations 2017 (British Institute of Radiology, 2015). All 
DEXA procedures will be conducted in accordance with IRMER standards. The protocol has 
been reviewed by a Medical Physics Expert (MPE) and a Clinical Radiation Expert (CRE), who 
have confirmed the justification, optimisation, and safety of the proposed radiation exposure. 
Supporting documentation has been submitted as part of the LREC application. 
 
14.3 Protocol compliance 
The trial will be conducted in full compliance with the approved protocol. No deviations will be 
made without prior approval, unless necessary to eliminate an immediate hazard to a 
participant. In the event of a medical emergency requiring a protocol deviation, the PI will use 
clinical judgment to protect the participant's safety and will promptly inform the supervisory team 
of the incident. If, in their judgment, the deviation warrants further action, the matter will be 
escalated to the LREC in accordance with institutional reporting requirements. All such 
decisions and actions will be documented and retained in the TMF. 
 
14.4 Notification of Serious Breaches to GCP and/or the protocol 
Serious breaches will be managed in accordance with regulatory requirements and LBU policy. 
A “serious breach” is defined as a breach likely to significantly affect either the safety or 
physical/mental integrity of trial participants, or the scientific value of the trial. In the event of a 
suspected serious breach during the conduct of the study, the academic supervisory team will 
be notified immediately. The supervisory team will assess the breach and, where applicable, 
notify the LREC in writing within seven calendar days of becoming aware of the incident. 
Notification will be made if the breach concerns either the conditions and principles of GCP or 
the approved trial protocol, including any amendments. Documentation of the breach, 
assessment, and subsequent actions will be maintained in the TMF. 
 
14.5 Data protection and patient confidentiality 
The PI and all designated research staff will comply with all applicable data protection laws, 
including the UK GDPR and the Data Protection Act 2018. The research team will implement 

 
 
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and maintain appropriate technical and organisational measures to protect personal data 
against unauthorised or unlawful processing, and against accidental loss, destruction, damage, 
alteration, or disclosure. 
 
All research team members involved in the processing of personal data will have completed 
mandatory data protection training as required by LBU. They will be bound by institutional 
confidentiality agreements or statutory obligations of confidentiality and will only process 
personal data as necessary for the conduct of the study. 
 
Personal data will be stored securely on LBU's password-protected servers or encrypted drives, 
with access limited to authorised members of the research team. Any physical documents (e.g. 
signed ICFs) will be stored in locked cabinets in secure locations on university premises. 
 
14.5.1 Subject data’ rights 
Participants will be informed of their data protection rights in the PIS, including their right to 
access, rectify, or request deletion of their data. No personal data will be transferred outside the 
UK without prior approval from the University and in full compliance with data protection 
legislation. The investigator or designee will implement and maintain appropriate technical and 
organizational measures to ensure accessibility to subjects of their personal data and their 
results of biological analysis collected during the study. Upon request, the investigator or 
designee will ensure to provide extracts of personal data to subjects. The investigator or 
designee will implement appropriate procedures to immediately inform the project supervisor 
about any subject’s request to rectify or delete their personal data or biological samples during 
the course of the trial. 
 
14.5.2 Confidentiality 
The PI will ensure protection of subject’s personal data and that all reports, publications, subject 
samples and any other disclosures, except where required by laws. Subjects are identified only 
by a subject identification number to maintain subject confidentiality. All subject trial records will 
be kept safely in an access-controlled area. Identification code lists linking subject names to 
subject identification numbers will be stored separate from subject records. In case of data 
transfer, the study team will maintain high standards of confidentiality and protection of subject 
personal data. Clinical information will not be released without the written permission of the 
subject, except for monitoring by Regulatory Authorities or the trial Sponsor. 
 
The PI will not engage any external third party to process personal data on behalf of the project 
without formal approval from LBU and without ensuring that appropriate data processing 
agreements are in place. 
 
14.5.3 Data Breaches reporting 
In the event of an actual or suspected data breach, including any unauthorised access to, or 
loss, disclosure, or destruction of personal data, the PI must report the incident to the 
University’s Data Protection Officer within 24 hours by calling 0113 812 7542 or emailing 
DPO@leedsbeckett.ac.uk. The research team will cooperate fully with the University’s breach 
response protocol, including any required actions to mitigate harm, restore data, and prevent 
recurrence. 
Where the PI or research team receives any request, complaint, or inquiry related to data 

 
 
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protection, such as subject access requests or concerns about data handling, this must be 
referred immediately to the University’s Data Protection Officer. 
 
14.5.4 Retention of Data 
The PI will maintain adequate trial records including (e)CRFs, laboratory reports, original signed 
ICFs, investigational product disposition records, safety reports, information regarding subjects 
who discontinued, and other pertinent data such as correspondence with the LREC and 
administrative documents exchanged with the research team. Records may be stored in either 
paper or electronic format. The PI must retain all trial records for five years, the maximum period 
required by LBU, unless otherwise notified. 
Biological samples collected during the trial will be retained for one year unless participants 
have agreed to a longer storage period. 
 
To avoid any possible errors, the PI must contact the project supervisor prior to the destruction 
of any trial records. The PI must also notify the project supervisor immediately in the event of 
accidental loss or destruction of any trial records. 
 
14.6 Financial and other competing interests 
None 
 
14.7 Indemnity 
This trial is sponsored by LBU, which holds appropriate insurance to cover liabilities arising from 
the design and management of the study, provided the protocol is followed. The PI is covered 
under the institution’s professional indemnity and clinical trials insurance. This includes cover for 
negligent harm arising from the conduct of the trial, where standard procedures are followed. 
 
14.8 Amendments 
Any permanent changes to the protocol will be formalized in an amended protocol which must 
be approved by the Research supervisor and, if substantial as defined in directive 2001/20/EC, 
submitted for approval to the LREC, prior to implementation. If the amendment results in a 
modification of trial treatment or subject assessments, a new version of the informed consent 
must be prepared and submitted for approval to the IRB. All protocol amendments will be 
documented and tracked in the protocol amendment log (Appendix B).  
 
14.9 Post trial care 
As the investigational product is already commercially available, participants will not receive 
continued access to the product from the research team after the trial concludes. However, 
participants will be informed, both during the informed consent process and at the end of the 
study, about how and where they can purchase the product should they wish to continue its use, 
based on discussions with their healthcare provider. 
No additional funding arrangements are in place for post-trial provision of the investigational 
product. All participants will return to standard NHS care following trial participation. Should any 
clinically relevant findings emerge during or after the trial that may affect participants’ ongoing 
care, they and their healthcare providers will be appropriately informed. 
 

 
 
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14.10 Access to the final trial dataset 
Access to the full trial dataset will be restricted to the PI and their academic supervisors at LBU, 
who are responsible for the design, conduct, and analysis of the study. These individuals will 
oversee data integrity, statistical analysis, and reporting. 
As this is a single-centre academic study, no formal restrictions apply to site-specific data 
access. However, should any additional investigators or collaborators be identified later in the 
trial, access to the full dataset will be granted only with documented approval from the Chief 
Investigator and academic supervisory team, following submission of a formal request outlining 
the intended use of the data. 
 
15 DISSEMINIATION POLICY 
 
15.1 Dissemination policy 
The data arising from this trial will be owned by the Sponsor, LBU. Upon completion of the trial, 
the data will be cleaned, analysed, and tabulated. A Final Trial Report will be prepared by the PI 
under the supervision of the academic supervisory team. This report will form part of the PI’s 
PhD thesis and may also contribute to peer-reviewed publications and conference 
presentations.  
 
Cultech, the commercial partner in this study, will have publication rights and will be 
acknowledged in all outputs arising from the trial. All publications and public dissemination of 
trial data will be shared with Cultech prior to submission, to allow for review and comment, but 
not to restrict or unduly delay publication. LBU and Cultech will collaborate to ensure timely and 
accurate communication of results. 
 
Participating investigators will not have independent rights to publish trial data without the 
written consent of the Chief Investigator and academic supervisory team. Any publication 
requests will be reviewed to ensure they do not compromise the integrity or timing of the main 
study results. There are no embargo periods or restrictions currently in place, though the 
primary publication will precede any secondary analyses. 
 
Publications will adhere to the CONSORT guidelines and checklist to ensure that reporting 
meets the standards required for submission to high-quality peer-reviewed journals 
(http://www.consort-statement.org/). 
 
Participants will be informed of the overall results of the trial after the main findings have been 
published or the Final Trial Report completed. This may be achieved through a lay summary 
disseminated via a newsletter, email, or study website, depending on participant preferences. 
Participants will also have the option to request a copy of the results from the PI. Individual-level 
results will not be disclosed, but summaries of study findings will be available on request after 
final analysis. 
 
Subject to ethical and data protection considerations, the trial protocol, anonymised participant-
level dataset, and statistical analysis code may be made publicly available following publication 
of the main findings. These materials will be hosted in the open-access LBU Research Data 
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sharing agreement, and details will be included in the published article. 
 
15.2 Authorship eligibility guidelines and any intended use of professional writers 
Authorship of the final trial report and any resulting publications will be determined in 
accordance with the criteria established by the International Committee of Medical Journal 
Editors (ICMJE). Individuals will be granted authorship if they have made substantial 
contributions to: (1) the conception or design of the study, or the acquisition, analysis, or 
interpretation of data; (2) drafting the manuscript or revising it critically for important intellectual 
content; (3) final approval of the version to be published; and (4) agreement to be accountable 
for all aspects of the work and its integrity. 
 
The PI will serve as lead author on the final trial report and primary publications, under the 
supervision of academic supervisors at LBU, who will also qualify for authorship based on their 
involvement in study design, oversight, and analysis. Representatives from Cultech may be 
included as co-authors where they meet the ICMJE criteria. All contributors who do not meet 
these criteria will be acknowledged appropriately in line with journal policies. Group authorship 
(e.g., on behalf of the Trial Team) may be used where appropriate and will be determined in 
advance of manuscript submission, particularly if additional collaborators contribute 
meaningfully to the conduct of the trial. 
 
No professional medical writers will be employed for this trial. Should this change, the 
involvement and funding source for any professional writing support will be transparently 
disclosed in the relevant publications.  
 
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17 APPENDICIES 
 
17.1 Appendix A: Schedule of Assessments 
 
 
Timepoint 
-1 
0 
1 
2 
3 
4 
 
 
Week 
-4 to-1 
-1 
0 
6 
11 12 
 
 
Window 
(days) 
≤30 
before 
screening 
0 
±7  
±7 
 
±7 
 
±7 
Activity 
Estimated 
duration 
(minutes) 
 
 
 
 
 
 
 
Pre-screen 
5 
 
X 
 
 
 
 
 
Informed Consent 
10 
 
 
X 
 
 
 
 
Eligibility Screen 
5 
 
 
X 
 
 
 
 
Randomisation 
N/A 
 
 
 
X 
 
 
 
Cognitive Testing 
Familiarisation 
20 
 
 
X 
 
 
 
 
Anthropometric 
Measurements 
5 
 
 
X 
 
X 
X 
 
Blood Pressure 
Assessment 
2 
 
 
X 
 
X 
X 
 
Fasting Blood Glucose 
Test 
2 
 
 
X 
 
X 
X 
 
HbA1c Testing 
2 
 
 
X 
 
X 
X 
 
Lipid Assessment 
2 
 
 
X 
 
X 
X 
 
Fasting Venous Blood 
Collection 
4 
 
 
X 
 
X 
X 
 
Oral Glucose 
Tolerance Test 
120 
 
 
X 
 
 
X 
 
PSQI 
3 
 
 
 
 
 
 
X 
Demographics and 
lifestyle Questionnaire 
6 
 
 
 
X 
 
 
 

 
 
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Dietary Habit Diary 
N/A 
 
 
 
X 
 
 
X 
Cognitive Testing 
(CANTAB) 
40 
 
 
 
X 
X 
 
X 
Doppler Ultrasound 
FMD and CVR 
35 
 
 
 
X 
 
 
X 
DXA Scanning 
15 
 
 
 
X 
 
 
X 
Stool Sample 
Collection 
N/A 
 
 
 
X 
 
 
X 
Health events and 
physical experiences 
questionnaire 
3 
 
 
 
 
X 
X 
X 
Adherence check 
N/A 
 
 
 
 
X 
X 
X 
Participant 
Compensation 
N/A 
 
 
 
 
 
 
X 
 
17.2 Appendix B: Amendment History 
Amendment 
No. 
Protocol 
version 
No. 
Date 
issued 
Author(s) 
of 
changes 
Details of changes made 
1. 
1.1 
28/08/
2025 
Lewis 
Hepburn 
Midpoint assessment revised from an 
online virtual visit to an in-person laboratory 
visit. Additional procedures introduced, 
including anthropometric measurements 
(height, weight, waist/hip circumference), 
cardiometabolic assessments (resting 
blood pressure, fasting capillary blood 
samples for glucose, HbA1c, and lipid 
profile), and venous blood collection. All 
relevant sections updates. 
Details of stool sample processing updated 
to reflect use of FTA card sampling.  
2.  
1.2  
20/03/
2026 
Lewis 
Hepburn 
The eligibility criteria have been amended 
to expand the upper age limit for participant 
inclusion from 35 years to 40 years. The 
revised age range is therefore 18–40 years. 
This amendment was introduced to 
improve recruitment feasibility while 

 
 
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maintaining a study population that is 
appropriate for the research objectives. 
Individuals within this age range are 
generally considered to represent a 
metabolically healthy young adult 
population with relatively low prevalence of 
age-related metabolic disease, thereby 
preserving the scientific rationale for 
studying mechanisms in healthy adults. 
Expanding the upper age limit by five years 
is not expected to introduce meaningful 
differences in cognitive or metabolic 
physiology that would compromise 
interpretation of the study outcomes. The 
amendment therefore supports improved 
recruitment while maintaining alignment 
with the study’s scientific aims and target 
population. 
2.  
1.2  
20/03/
2026 
Lewis 
Hepburn 
The CVR protocol has been updated to use 
a standardized breath-hold paradigm in 
place of externally administered CO₂. 
Participants will complete a 1-minute 
normocapnic baseline followed by a 30-
second breath hold with nasal occlusion, 
with exhalation at the end of the maneuver 
to capture peak EtCO₂. Continuous MCA 
velocity and EtCO₂ will be recorded 
throughout. CVR will be calculated as the 
percent change in MCA velocity from 
baseline to peak divided by the change in 
EtCO₂ (%/mmHg). 
This modification improves feasibility and 
tolerability while maintaining a 
physiologically valid hypercapnic stimulus 
and aligning the analysis approach with the 
stimulus. 

 
 
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2.  
1.2  
20/03/
2026 
Lewis 
Hepburn 
Addition of platelet activity as an 
exploratory outcome, assessed using 
venous blood samples already being 
collected. No changes to participant 
procedures, burden, or risk. For 
participants already enrolled, only those 
who have provided explicit consent for 
additional analyses of their blood samples 
will be included; those who have not will be 
excluded from this component. All newly 
recruited participants will be consented for 
this analysis as part of standard study 
procedures.