The Impact of Type 2 Diabetes and Exercise on Liver Fat Quality
Phase: NA
Conditions: Type 2 Diabetes Mellitus, Non-Alcoholic Fatty Liver Disease, Obesity, Insulin Resistance, Liver Diseases, Fatty Liver, Diabetes Mellitus, Glucose Metabolism Disorders, Glucose Intolerance, Metabolic Disease
Sponsor: James King

 
 
 
 
 
 
 
 
 
 
Full title 
 
 
The Impact of Type 2 Diabetes and Exercise on Liver Fat Quality 
 
 
Short title 
 
 
Diabetes, Exercise and Liver Fat (DELIVER) 
 
 
 
 
 
 
 
Confidentiality Statement 
All information contained within this protocol is regarded as, and must be kept, confidential. No part of it 
may be disclosed by any Receiving Party to any Third Party, at any time, or in any form without the 
express written permission from the Chief Investigator and/or Sponsor. 
 
 
 
 
 
 
 
 
 
 
 
 
 
i 
 
Research protocol version 6 
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The Impact of Type 2 Diabetes and Exercise on Liver Fat Quality 
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Chief Investigator: 
Dr James King 
Contact Details: 
Institution: Loughborough University 
School: Sport, Exercise & Health Sciences 
Address: Epinal Way 
Loughborough 
Leicestershire 
UK 
LE11 3TU 
Email: J.A.King@lboro.ac.uk 
Investigators: 
Professor Guruprasad Aithal 
Professor Melanie Davies 
Professor Penny Gowland 
Professor David Stensel 
Dr Tom Yates 
Dr David Webb 
Dr Stephen Bawden 
Dr Ghazala Waheed 
Mr Jack Sargeant 
Mr Scott Willis 
Mrs Sundus Malaikah 
Sponsor: 
Loughborough University 
Ethics Ref (IRAS Project ID): 241046 
Funders: 
Diabetes UK (ref no. 17/0005659); NIHR Nottingham & 
Leicester Biomedical Research Centres 
23/09/21 
Version 6 
 
Date and Version No: 

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Authors 
 
 
1. Dr James King 
2. Professor Guruprasad Aithal 
3. Dr David Webb 
4. Professor Melanie Davies 
5. Professor Penny Gowland 
6. Professor David Stensel 
7. Dr Tom Yates 
8. Dr Stephen Bawden 
9. Dr Ghazala Waheed 
10. Mr Jack Sargeant 
11. Mr Scott Willis 
12. Mr Aron Sherry 
13. Mrs Sundus Malaikah 

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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 study in compliance with the approved protocol and will adhere to the 
principles outlined in the Declaration of Helsinki, the Sponsor’s SOPs, and other regulatory 
requirements. 
 
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 investigation without the prior written consent 
of the Sponsor. 
 
I also confirm that I will make the findings of the study publicly available through publication or other 
dissemination tools without any unnecessary delay and that an honest accurate and transparent account 
of the study will be given; and that any discrepancies from the study as planned in this protocol will be 
explained. 
 
 
Chief Investigator: 
Date: 
23/09/2021 
Name: Dr JAMES KING 

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LIST of CONTENTS 
 
SECTION 
Page No. 
TITLE PAGE 
i 
RESEARCH REFERENCE NUMBERS 
ii 
AUTHOR LIST 
iii 
SIGNATURE PAGE 
iii 
LIST OF CONTENTS 
iv 
1 
AMENDMENT HISTORY 
1 
2 
STUDY CONTACTS 
2 
3 
STUDY SUMMARY 
8 
4 
FUNDING AND SUPPORT IN-KIND 
10 
5 
ABBREVIATIONS 
12 
6 
PEER REVIEW 
13 
7 
STUDY BACKGROUND AND RATIONALE 
14 
8 
OBJECTIVES 
16 
9 
STUDY DESIGN 
18 
10 
TRIAL PARTICIPANTS 
22 
11 
STUDY PROCEDURES 
25 
12 
TREATMENT OF TRIAL PARTICIPANTS 
29 
13 
SAFETY REPORTING 
33 
14 
STATISTICS 
36 
15 
ACCESS TO SOURCE DATA / DOCUMENTS 
38 
16 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
39 
17 
CODE OF PRACTICE AND REGULATIONS 
40 
18 
DATA HANDLING AND RECORD KEEPING 
42 
19 
STUDY GOVERNANCE 
43 
20 
FINANCE AND INSURANCE 
44 
21 
PUBLICATION / DISSEMINATION POLICY 
45 
22 
REFERENCES 
46 

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1. AMENDMENT HISTORY 
 
 
Protocol version 1 – 1/5/2018 
Protocol version 2 – 14/11/2018 
Protocol version 3 – 19/02/2020 
Protocol version 4 – 20/05/2020 
Protocol version 5 – 09/09/2020 
Protocol version 6 – 23/09/2021 
 
 
 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Author(s) of 
changes 
Details of Changes made 
2 
2 
14/11/18 
James King, Aron 
Sherry 
In our protocol, in relation to pre-exercise screening, we 
have used the term ‘medical professional’ which should 
be ‘healthcare professional’. The subtle change in 
terminology clarifies that the individual responsible for 
the pre-exercise assessment is not necessarily a doctor 
but can be another suitably qualified healthcare 
professional – in our case a specialist cardiac nurse 
3 
2 
14/11/18 
James King, Aron 
Sherry 
The ‘medical exam’ or ‘physical exam’ referred to in 
protocol v1 is actually an optional part of the Leicester 
BRC’s pre-exercise screening process. When writing the 
protocol we misinterpreted the BRC pre-exercise 
screening SOP (that all medical evaluations include a 
physical exam). In effect, our SOP states that all 
participants undergo a medical history, resting BP and 
12-lead ECG; but a physical exam only occurs if the 
healthcare professional dictates it is necessary – based 
on the history, ECG and BP. If the physical exam is 
needed, this is performed by a doctor. 
4 
3 
19/02/20 
James King, Aron 
Sherry, Scott 
Willis 
In protocol v2, a participant inclusion criterion 
stipulated “Inactive (low or medium score on IPAQ- 
short form)” and an exclusion criterion stipulated 
“Excessively active (score of high on IPAQ-short 
form)”. These items have now been removed from the 
eligibility criteria and the IPAQ-short form has been 
removed as a screening tool. Instead, within the 
exclusion criteria, an item has been added which 
excludes individuals who regularly take part in 
purposeful exercise training of vigorous intensity. This 
change was to more effectively screen out individuals 
who are too physically fit for the purpose of the study. 
5 
4 
20/05/2020 
James King, Scott 
Willis 
The inclusion criteria for Part B of the study has now 
been changed to the recruitment of both patients with 
pre-diabetes and type 2 diabetes. The involved reducing 
the inclusion criterion for HbA1c from 6.5% to 6.0% 
and changing references to this group as ‘individuals 
with type 2 diabetes/prediabetes’. The change was to 
enable a wider pool of patients to be recruited from in 
order to complete the study in a timely fashion due to 
ongoing difficulties with patient recruitment. Mrs 
Sundus Malaikah has also been added as a co-
investigator on the study. 

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6 
5 
09/09/2020 
James King, Scott 
Willis 
The requirement of participants to complete three 
supervised and one unsupervised exercise training 
session per week has been altered so that participants 
are now required to complete four exercise training 
sessions per week, of which at least one session will be 
supervised by a member of the research team. This 
change is to reduce the number of visits participants 
make to study sites in order to reduce potential exposure 
to COVID-19. Participants will also now be contacted 
via telephone after each unsupervised session for 
support and to ensure compliance with the prescribed 
exercise intensity and duration. Site-specific COVID-19 
symptom screening questionnaires have also been added 
which will be administered verbally by a member of the 
research team within 72 hours of each visit to study sites 
and on the day of each visit.  
7 
6 
23/09/2021 
James King, Scott 
Willis 
The expiration date for the NHS ethical approval was 
extended from 31/12/2021 to 31/12/2022. This was in 
response to an 18 month delay due to the COVID-19 
pandemic to allow more time for participant recruitment 
and data collection to hit the study recruitment targets. 

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2. KEY STUDY CONTACTS 
 
Chief Investigator 
Dr James King 
Lecturer in Exercise Physiology 
School of Sport, Exercise & Health Sciences 
Loughborough University 
Loughborough 
Leicestershire 
UK 
LE11 3TU 
Phone: 01509 228457 
Email: J.A.King@lboro.ac.uk 
Note: postal correspondence to be directed to the Sir 
John Beckwith Building, Loughborough University, 
Leicestershire, UK, LE11 3TU 
Study Co-ordinator 
The Chief Investigator will co-ordinate the study 
Sponsor 
Loughborough University, Leicestershire, UK 
Contact: 
Dr Jen Fensome 
Director of Research and Enterprise 
Research & Enterprise Office 
Hazlerigg Building Loughborough University 
Leicestershire 
UK 
LE11 3TU 
Phone: 01509 228693 
Fax: 01509 223953 
Email: j.fensome@lboro.ac.uk 
Funders 
Diabetes UK 
Wells Lawrence House 
126 Back Church Lane 
London 
E1 1FH 
Phone: 0345 123 2399 

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Email: info@diabetes.org 
Fax: 020 7424 1001 
The NIHR Leicester Biomedical Research Centre 
Contact: Mr Tim Skelton (BRC manager) 
Diabetes Research Centre 
Leicester General Hospital 
Gwendolen Road 
Leicester 
LE5 4PW 
Phone: 0116 258 8128 
Email: tim.skelton@uhl-tr.nhs.uk 
The NIHR Nottingham Biomedical Research Centre 
Contact: Prof Guruprasad Aithal (Liver Theme Lead) 
Queens Medical Centre Hospital, West Block, E floor, Room 
1418 
Nottingham 
UK 
NG7 2UH 
Phone: 0115 823 1149 
Email: Guru.Aithal@nottingham.ac.uk 
Fax: 0115 970 9012 
Protocol authors / co-investigators 
Dr James King (CI & PI – Loughborough) 
Lecturer in Exercise Physiology 
School of Sport, Exercise & Health Sciences 
Loughborough University 
Loughborough 
Leicestershire 
UK 
LE11 3TU 
Phone: 01509 228457 
Email: J.A.King@lboro.ac.uk 

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Professor Guruprasad Aithal (PI – Nottingham) 
Professor of Hepatology 
Queens Medical Centre Hospital, West Block, E floor, Room 
1418 
University of Nottingham 
Nottingham 
UK 
NG7 2UH 
Phone: 0115 823 1149 
Email: Guru.Aithal@nottingham.ac.uk 
Dr David Webb (PI – Leicester) 
Clinical Senior Lecturer in Diabetes Medicine 
Diabetes Research Centre 
University of Leicester 
Leicester General Hospital 
Gwendolen Road 
Leicester 
UK 
LE5 4PW 
Phone: 0116 258 8554 
Email: david.webb@uhl-tr.nhs.uk 
Professor Melanie Davies 
Professor of Diabetes Medicine 
Diabetes Research Centre 
University of Leicester 
Leicester General Hospital 
Gwendolen Road 
Leicester 
UK 
LE5 4PW 
Phone: 0116 258 6481 
Email: melanie.davies@uhl-tr.nhs.uk 

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Professor Penny Gowland 
Professor of Physics 
Sir Peter Mansfield Imaging Centre (room MR19) 
University Park 
Nottingham 
UK 
NG7 2RD 
Phone: 0115 951 4754 
Email: penny.gowland@nottingham.ac.uk 
Dr Thomas Yates 
Reader in Physical Activity, Sedentary Behaviour and Health 
Diabetes Research Centre 
University of Leicester 
Leicester General Hospital 
Gwendolen Road 
Leicester 
UK 
LE5 4PW 
Phone: 0116 258 4312 
Email: ty20@leicester.ac.uk 
Professor David Stensel 
Professor of Exercise Metabolism 
School of Sport, Exercise & Health Sciences 
Loughborough University 
Loughborough 
Leicestershire 
UK 
LE11 3TU 
Phone: 01509 226344 
Email: D.J.Stensel@lboro.ac.uk 
Mr Jack Sargeant 
Research Associate 

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Diabetes Research Centre 
University of Leicester 
Leicester General Hospital 
Gwendolen Road 
Leicester 
UK 
LE5 4PW 
Phone: 0116 258 4389 
Email: js928@le.ac.uk 
Dr Stephen Bawden 
Research Associate 
Sir Peter Mansfield Imaging Centre 
University Park 
Nottingham 
UK 
NG7 2RD 
Phone: 01159 514747 
Email: Stephen.Bawden@nottingham.ac.uk 
Dr Ghazala Waheed 
Clinical Trial Statistician 
Diabetes Research Centre 
University of Leicester 
Leicester General Hospital 
Gwendolen Road 
Leicester 
UK 
LE5 4PW 
Email: gw136@leicester.ac.uk 
Mr Scott Willis  
PhD Student 
School of Sport, Exercise & Health Sciences 
Loughborough University 

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Loughborough 
Leicestershire 
Phone: 07950 938338 
Email: s.willis2@lboro.ac.uk 
Mr Aron Sherry 
Research Associate NIHR Leicester BRC 
National Centre for Sport & Exercise Medicine 
Loughborough University 
Leicestershire 
UK 
LE11 3TU 
Email: A.P.Sherry@lboro.ac.uk 
 
Mrs Sundus Malaikah 
PhD Student 
School of Sport, Exercise and Health Sciences 
Loughborough University  
Loughborough 
Leicestershire 
Phone: 07940 013742 
Email: S.Malaikah@lboro.ac.uk 
 
Committees 
Trial Steering Committee 
Contact: 
Dr James King (Chair) 
Lecturer in Exercise Physiology 
School of Sport, Exercise & Health Sciences 
Loughborough University 
Loughborough 
Leicestershire 
UK 
LE11 3TU 
Phone: 01509 228457 
Email: J.A.King@lboro.ac.uk 

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3. STUDY SUMMARY 
 
Full study title 
The Impact of Type 2 Diabetes and Exercise on Liver Fat 
Quality 
Internal ref. no. (or short title) 
Diabetes, Exercise & Liver Fat (DELIVER) 
Study Design 
Part A: cross-sectional study 
 
Part B: randomised controlled trial (RCT) 
Scientific abstract 
Non-alcoholic fatty liver disease (NAFLD) is characterised by 
an excessive accumulation of liver fat which is intricately linked 
to type 2 diabetes (T2DM), cardiovascular and renal disease. 
Recent research suggests that liver fat quality i.e. the 
composition of fat stored in the liver (saturated, unsaturated, 
polyunsaturated), may represent a key link between excess 
liver fat and adverse metabolic health outcomes. The proposed 
research contains a cross-sectional component (Part A) that 
will explore whether liver fat quality differs in male NAFLD 
patients with (n = 26) or without T2DM (n = 14); and a 
randomised controlled trial (Part B) (one exercise training and 
one control group) that will determine if exercise (150 - 200 min 
per week, 6 weeks) can beneficially modify liver fat quality in 
NAFLD patients with T2DM/prediabetes (n = 26, 13 per group). 
Liver fat quality will be assessed in both studies via magnetic 
resonance (3T) spectroscopy (1H-MRS) using validated 
methods. This research will investigate whether liver fat quality 
is a novel therapeutic target for T2DM patients with NAFLD and 
determine whether exercise is able to favourably modify liver 
fat quality. 
Study Participants 
Part A (cross-sectional study): 14 obese men with non- 
alcoholic fatty liver disease (NAFLD) and 26 obese men with 
NAFLD and T2DM 
Part 
B 
(RCT): 
26 
obese 
men 
with 
NAFLD 
and 
T2DM/prediabetes 
Planned Study Period 
1st September 2018 to 31st December 2022 (52 months) 

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Research aims and objectives 
The research outlined in this proposal seeks to determine the 
impact of T2DM on liver fat quality in patients with NAFLD and 
to characterise changes in liver fat quality in response to a 
supervised exercise training programme. 
The specific objectives of this work are to: 
 
• characterise 
the 
liver 
fat 
phenotype 
(saturation, 
unsaturation and polyunsaturation indices) and associated 
metabolic parameters in obese men with NAFLD and T2DM 
versus non-diabetic obese men with NAFLD 
• determine the impact of six weeks supervised exercise 
training on liver fat quality (saturation, unsaturation and 
polyunsaturation indices) and associated metabolic 
parameters in obese men with NAFLD and 
T2DM/prediabetes. 

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4. FUNDING AND SUPPORT IN KIND 
 
FUNDER(S) 
FINANCIAL AND NON-FINANCIAL 
SUPPORT GIVEN 
Diabetes UK 
Wells Lawrence House 
126 Back Church Lane 
London 
E1 1FH 
Phone: 0345 123 2399 
Email: info@diabetes.org 
Fax: 020 7424 1001 
Early Career Small Grant (Dr James King - Chief 
Investigator) - £15,000 
The NIHR Nottingham Biomedical Research 
Centre 
Contact: Prof Guruprasad Aithal (Liver Theme 
Lead) 
Queens Medical Centre Hospital, West Block, E 
floor, Room 1418 
Nottingham 
UK 
NG7 2UH 
Phone: 0115 823 1149 
Email: Guru.Aithal@nottingham.ac.uk 
Fax: 0115 970 9012 
• Funding support for additional MRI scans 
(see breakdown in section 20) 
• Clinical Research Fellow support 
The NIHR Leicester Biomedical Research 
Centre 
Contact: Mr Tim Skelton (BRC manager) 
Diabetes Research Centre 
Leicester General Hospital 
• Funding support for travel expenses 
(participants and study investigators) and 
metabolic analyses (see breakdown in 
section 20) 
• Research Associate (0.5 WTE, 2 years) 
• PhD student 

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Gwendolen Road 
Leicester 
LE5 4PW 
Phone: 0116 258 8128 
Email: tim.skelton@uhl-tr.nhs.uk 
• Research Administrator 
• Cardiac Nurse 
• Clinical Research Fellows 
• Leicester Diabetes Centre Exercise Team 

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5. 
ABBREVIATIONS 
 
 
AE 
Adverse event 
AR 
Adverse reaction 
BMI 
Body Mass Index 
BRC 
NIHR Biomedical Research Centre 
CI 
Chief Investigator 
CRF 
Case Report Form 
CRN 
Clinical Research Network 
CRO 
Contract Research Organisation 
CT 
Clinical Trials 
EC 
Ethics Committee (see REC) 
GCP 
Good Clinical Practice 
GP 
General Practitioner 
1H-MRS 
Protein Magnetic Resonance Spectroscopy 
ICF 
Informed Consent Form 
IRAS 
Integrated Research Application System 
MRI 
Magnetic Resonance Imaging 
NAFLD 
Non-Alcoholic Fatty Liver Disease 
NHS 
National Health Service 
NRES 
National Research Ethics Service 
PI 
Principal Investigator 
PIS 
Participant/ Patient Information Sheet 
PIL 
Participant/ Patient Information Letter 
R&D 
NHS Trust R&D Department 
REC 
Research Ethics Committee 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SD 
Standard Deviation 
SOP 
Standard Operating Procedure 
SUSAR 
Suspected Unexpected Serious Adverse Reactions 
TMF 
Trial Master File 
TSC 
Trial Steering Committee 
T2DM 
Type 2 Diabetes Mellitus 

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6. PEER REVIEW 
 
 
The research outlined in this protocol document has been peer reviewed by four independent experts in 
the research field during the Diabetes UK grant award process (competitive funding process). Within 
this process, the chief investigator was required to amend or rebut reviewer’s comments and the final 
protocol has therefore been refined based on this critique. A diabetes-focussed PPI group at the 
Leicester General Hospital (organised by the NIHR Leicester BRC) also provided detailed comments on 
this research study during the development of the Diabetes UK funding application. 

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7. BACKGROUND AND RATIONALE 
 
Non-alcoholic fatty liver disease (NAFLD) is often described as the hepatic manifestation of the 
metabolic syndrome and is defined as the excessive accumulation of fat within the liver (hepatic 
steatosis) in individuals who do not consume excessive alcohol or possess viral hepatic pathology [1]. 
The global prevalence of NAFLD is high and rising owing to the pandemics of obesity and inactivity 
which are leading risk factors. The most recent estimates suggest that NAFLD is present in 
approximately 25% of adults and up to 75% of those who are obese [2]. These statistics are concerning 
given that changes in metabolism that occur with NAFLD are heavily implicated in the development and 
progression of type 2 diabetes (T2DM), cardiovascular and renal disease [3]. 
 
The pathophysiology of NAFLD is complex and not completely understood; however, it is recognised 
that insulin resistance is integral to disease initiation and progression [4]. In effect, hepatic steatosis is 
strongly associated with insulin resistance in the liver, skeletal muscle and adipose tissue (body fat) [5]. 
This pathology helps explain the high prevalence of NAFLD in individuals with T2DM (up to 90%) and 
the more aggressive trajectory witnessed in this patient group [6]. Notably, approximately 40% of T2DM 
patients with NAFLD have non-alcoholic steatohepatitis (NASH) [6]; a more progressive form of NAFLD 
characterised by liver inflammation and injury that predisposes to premature cardiovascular and liver- 
related mortality [7]. This knowledge underlines the need to identify effective methods to prevent the 
inception and progression of NAFLD for those with, and at risk of, T2DM. 
 
Although there is a strong link between the liver inflammation and injury associated with NASH and 
adverse clinical consequences; including substantially higher rates of decompensated cirrhosis, liver 
cancer, all-cause mortality and cardiovascular events [8], no such relationship exists between the 
amount of liver fat and clinical outcomes. However, recent research has identified liver fat quality (lipid 
species proportions) as being central to the metabolic derangements associated with NAFLD; with a 
higher proportion of saturated lipids, and a lower proportion of unsaturated lipids, being linked to adverse 
metabolic phenotypes e.g. insulin resistance and dyslipidaemia [9,10]. It is possible that elevated 
proportions of saturated hepatic lipids may contribute to the more aggressive NAFLD trajectory seen in 
patients with T2DM. This hypothesis is supported by the knowledge that hepatic de novo lipogenesis; a 
significant contributor to the hepatic fat pool in NAFLD [11]; exclusively produces saturated lipids [12] 
and is stimulated directly by hyperinsulinemia and hyperglycemia [12]. Liver fat quality may therefore 
represent a new therapeutic target for patients with NAFLD and T2DM. 
The importance of liver fat quality within the pathophysiology of NAFLD has been identified within studies 
that have analysed liver tissue specimens collected via surgical biopsy [9,10]. Liver biopsy is a highly 
invasive procedure that carries significant risks to the patient. Alternatively, a MRI based procedure has 
been validated as a technique that is able to non-invasively determine liver fat quality indices (saturated, 

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unsaturated and polyunsaturated indexes) [13]. In this original paper, several lipids of known 
composition were analysed via proton magnetic resonance spectroscopy and were shown to assess 
liver fat quality with a high degree of accuracy and precision. Additionally, the researchers also identified 
higher saturated fat and lower unsaturated fat indices in obese individuals with clinically elevated hepatic 
steatosis verses obese and lean individuals without elevated liver fat. The MRI technique detailed in this 
research study provides a useful tool for researchers to investigate the importance of liver fat quality in 
the pathophysiology of NAFLD (and associated disease) and to scrutinise the efficacy of disease 
management therapies. 
 
A small feasibility study in patients with NAFLD has demonstrated that seven consecutive days of 
moderate-intensity brisk walking is able to increase the proportion of polyunsaturated lipids within the 
liver and improve insulin sensitivity [14]. This beneficial change in liver fat quality occurred independently 
of body weight change and was directly associated with an increase in circulating adiponectin. These 
preliminary findings suggest that exercise may directly improve hepatic fat quality and metabolic 
outcomes in patients with NAFLD but further research is now required to investigate this possibility. It is 
intriguing that formal exercise training provides robust metabolic health benefits for patients with NAFLD, 
despite conferring a relatively modest weight-independent reduction in total liver fat [15,16]. This 
research project will explore the possibility that the quality of liver fat may contribute to the adverse 
metabolic profile seen in individuals with T2DM and NAFLD as well as examining the potential for regular 
exercise training to elicit favourable changes in liver fat quality. This research will therefore expand 
knowledge relating to the pathophysiology of NAFLD and T2DM; and the efficacy of exercise as a non- 
pharmacological therapy. 

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8. OBJECTIVES 
Co-primary objectives 
To determine: a) whether differences exist in the liver saturated fat index between obese men with 
NAFLD, with versus without, T2DM; b) the impact of six weeks exercise training on these outcomes in 
obese men with NAFLD and T2DM/prediabetes. 
 
Secondary objectives 
 
 
• 
To characterise potential differences in the following outcomes between obese men with 
NAFLD and T2DM versus non-diabetic obese men with NAFLD 
o Liver unsaturated fat index 
o Liver polyunsaturated fat index 
o Total liver fat percentage 
o Liver inflammation 
o Visceral adipose tissue 
o Circulating cardiometabolic biomarkers 
o Circulating liver enzymes 
o Circulating hepatokines 
o Objectively measured sedentary time and physical activity 
o Energy and macronutrient intake 
o Body weight & body fat percentage 
o Blood pressure 
o Aerobic fitness (peak oxygen uptake) 
• 
To determine the impact of six weeks moderate-intensity exercise training on the following 
outcomes in obese men with NAFLD and T2DM/prediabetes 
o Liver unsaturated fat index 
o Liver polyunsaturated fat index 
o Total liver fat percentage 
o Liver inflammation 
o Visceral adipose tissue 
o Circulating cardiometabolic biomarkers 
o Circulating liver enzymes 
o Circulating hepatokines 

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o Objectively measured sedentary time and habitual physical activity 
o Energy and macronutrient intake 
o Body weight and body fat parentage 
o Blood pressure 
o Aerobic fitness (peak oxygen uptake) 

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9. STUDY DESIGN 
Summary of trial design 
This study consists of two parts: a cross-sectional component (Part A) and a randomised controlled trial 
(RCT) (Part B). All participants will complete Part A (non-diabetic obese men with NAFLD and obese 
men with NAFLD & T2DM/prediabetes); however, only one of the two study groups will progress to Part 
B (obese men with NAFLD and T2DM/prediabetes) (see Figure 1). 
 
All prospective study participants will undergo a telephone interview in order to verbally assess potential 
study eligibility. This conversation will be conducted by a member of the study team and is anticipated 
to last up to 30 minutes. Participants wishing to take part and who appear eligible will subsequently be 
invited to attend the first face-to-face study assessment visit (visit one). 
 
Study assessment visit one will occur at the Sir Peter Mansfield Imaging Centre located on the University 
of Nottingham Campus. Written informed consent shall be obtained at the beginning of this visit. After 
this, several questionnaires relating to health status and lifestyle habits will be completed before 
anthropometric measurements are taken. Participants will then undergo an MRI scan of the liver and 
abdominal region. Before participants leave the centre they shall be familiarised with a wrist worn (watch-
like) physical activity monitor that they will wear continuously for the next seven days; and a food diary 
that they will complete on two week and one weekend day. 
 
Participants will be invited to attend a second assessment visit if their MRI scan shows that they meet 
the criteria for clinically elevated liver fat (> 5.56%). Visit two will take place at the Queens Medical 
Centre Hospital (Nottingham) or the Leicester General Hospital (depending on participant preference). 
During this session a fasting blood sample will be taken and a healthcare professional will perform a 
medical exam that will include an ECG. Finally, participants will complete a symptom limited peak oxygen 
uptake test on a treadmill. Vital signs will be monitored throughout this test by a healthcare professional. 
 
Participants’ involvement in this study will end after visit two for those only taking part in Part A. At this 
point, participants destined to progress to Part B of the study will be randomised to one of two six week 
interventions (1:1) (exercise training or control) by the trial statistician. Participants will be notified of 
their assignment by telephone. 
 
Participants randomised to control will receive no interventions and will be requested to maintain their 
habitual lifestyle during the six week intervention phase. Ahead of week five, participants will be posted 
a wrist worn physical activity monitor to wear continuously for the final two weeks of the study. 

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Participants will also complete a food diary during this time (two week and one weekend day in week 
five and six). 
 
Participants randomised to the exercise training intervention will complete 24 moderate-intensity 
exercise training sessions over the subsequent six weeks (four times per week; ~50 min per session). 
Each week, at least one of the four exercise training sessions will be supervised by the research team. 
The remainder of the sessions each week will be unsupervised but monitored objectively using a heart 
rate monitor. Post- intervention assessments will be completed 48 h - 72 h after the final exercise training 
session. 
 
After the intervention phase, participants will attend two final assessment sessions to obtain post- 
intervention data. Assessment visit three will be a repeat of assessment visit one whilst assessment visit 
four will be a repeat of assessment visit two (minus the medical examination). 

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Assessment visit 2 (QMC or LDC) 
Assessments 
Medical evaluation 
Blood sample 
VO2  peak test 
7 day physical activity monitor; 3 days food diary 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: flow diagram of the study protocol 
SPMIC – Sir Peter Mansfield Imaging Centre; QMC – Queens Medical Centre, Nottingham; LDC – 
Leicester Diabetes Centre; LU – Loughborough University 
Phone Interview (verbal eligibility) 
Prospective participants 
Part A (cross-sectional study) 
Assessment visit 1 (SPMIC) 
26 obese men with NAFLD & T2DM 
Consent 
Questionnaires 
Anthropometry, blood pressure, HbA1c 
MRI scan 
Assessment visit 1 (SPMIC) 
14 obese men with NAFLD 
Consent 
Questionnaires 
Anthropometry, blood pressure, HbA1c 
MRI scan 
Assessment visit 2 (QMC or LDC) 
Assessments 
• Medical evaluation 
• Blood sample 
• VO2  peak test 
Control 
(N = 13) 
 
No intervention 
Exercise 
Training 
(QMC/ LDC/LU 
(N = 13) 
4 x /week (3 
supervised) 
150 – 200 
min 
Part B (RCT) 
Randomisation 
Assessment visit 4 (QMC or LDC) 
• Blood sample 
• VO2  peak test 
Assessment visit 3 (SPMIC) 
• Anthropometry, blood pressure, 
HbA1c, MRI 

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Primary and secondary endpoints / outcome measures 
 
 
Co-primary outcomes: Liver saturated lipid index (%) determined by proton magnetic resonance 
spectroscopy (1H-MRS) using a 3T MRI scanner 
1) Comparison between obese men with NAFLD verses obese men with NAFLD and T2DM (Part  A) 
2) Response to the six week exercise training intervention (Part B) 
 
 
Secondary outcomes: 
• 
Liver unsaturated lipid index (%) - measured using 1H-MRS 
• 
Liver polyunsaturated lipid index (%) - measured using 1H-MRS 
• 
Total hepatic lipid composition (%) - measured using 1H-MRS 
• 
Liver inflammation - measured using MRI 
• 
Visceral adipose tissue - measured using MRI 
• 
Subcutaneous abdominal adipose tissue - measured using MRI 
• 
Body mass and body fat percentage 
• 
Blood pressure 
• 
Aerobic fitness (peak oxygen uptake) 
• 
Circulating metabolic biomarkers (fasted blood sample) 
o Liver enzymes 
o Glucose 
o Insulin 
o Lipids 
o HbA1c 
o HOMA-IR (surrogate marker of peripheral insulin resistance) 
o ADIPO-IR (surrogate marker of adipose tissue insulin resistance) 
o Inflammatory proteins 
o Hepatokines 
• 
Objectively measured sedentary time & physical activity assessed via the GENEactiv physical 
activity monitor 
• 
Energy and macronutrient intake (two weekdays and one weekend day) 

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10. TRIAL PARTICIPANTS 
Overall description of trial participants 
The participants we are seeking to recruit are inactive and obese men, with clinically elevated liver fat, 
with or without T2DM/prediabetes. The inclusion and exclusion criteria can be found overleaf. 
 
Recruitment strategy 
Recruitment will target individuals within multiple settings: hospital clinics, primary care, community 
weight management services, research databases (UHL & NUH NHS Hospital Trusts, Loughborough 
University), the community, internet and local media. 
 
Hospital clinics 
Within relevant diabetes, liver disease and bariatric surgery clinics across Leicester and Nottingham, 
study posters will be displayed, and clinicians may identify participants and distribute Participant 
Information Leaflets and Participant Information Sheets. 
 
General practice and primary care 
Across Leicestershire and Nottinghamshire, GP surgeries will be contacted to request support with 
recruitment for this study. If willing, they will be asked to identify potential participants and circulate 
Invitation Letters and Participant Information Sheets. Access to GP surgeries will be supported by the 
CRN and regional CCGs. Other primary care centres (dental practices, opticians, community 
pharmacies) within Nottingham University Hospitals NHS Trust and University Hospitals Leicester NHS 
Trust will also be asked to display advertisement materials (study poster) and provide Participant 
Information Leaflets. 
 
Community weight management services 
The study will be advertised by providers of Tier 2 (lifestyle intervention) and 3 (specialist weight 
management programs) weight management services within Leicestershire, Nottinghamshire and 
Derbyshire. Session leaders will identify potential participants via databases or during clinical contacts 
and provide copies of Participant Information Leaflets / Sheets. The study will also be advertised via 
poster at service facilities. 
 
Research databases 
Eligible individuals who have previously taken part in research studies and whom have consented to be 
contacted about future research studies will be contacted. Investigators involved in this study have 
access to databases of such individuals across the NIHR Nottingham BRC, NIHR Leicester BRC and 
Loughborough University. Potential participants will either be posted a Participant Information Leaflet or 

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contacted via telephone – depending on the preference of the investigator who is responsible for 
curating the participant database. 
 
Community and internet 
Participants will be recruited from the general public through various routes: 
• 
Commercial weight management programmes: session leaders from local weight loss programs 
will advertise the study to their cohorts by mentioning the study at meetings and providing copies 
of Participant Information Leaflets / Sheets. 
• 
Websites: the study will be advertised on University (Loughborough, Nottingham and Leicester) 
and Hospital Trust (Nottingham, Leicester) websites where links to investigator contact details 
and electronic versions of study information resources will be provided. Anyone interested in 
taking part will subsequently be able to contact the research team. 
• 
Local media: the research team will liaise with University (Loughborough, Nottingham and 
Leicester) public relations teams to advertise the study within the community. Advertisements 
routes may include press releases (website, radio, newspaper) where investigator’s contact 
details will be provided for anyone who may be interested in taking part after hearing about the 
study. 
• 
Study posters: will be displayed in hospital trusts, university and local community (where 
permitted). 
 
Inclusion criteria 
• 
Men 
• 
≥ 30 - ≤ 75 years of age 
• 
Body mass index ≥ 27 - ≤ 45 kg/m2 (or ≥ 23 to 45 45 kg/m2 if south Asian) 
• 
Waist circumference ≥ 94 cm (or ≥ 90 cm if south Asian) 
• 
Clinically elevated liver fat (≥ 5.56% assessed via 1H-MRS)[17] 
• 
Participant is willing and able to give informed consent to participate 
• 
Participant can communicate effectively in English 
• 
Participant is able to meet the time demands of the study 
 
 
Additional criteria for participants without T2DM 
• 
HbA1c < 6% 
Additional criteria for participants with T2DM/prediabetes 
• 
Diagnosed T2DM or prediabetes 

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• 
Treatment via lifestyle or metformin only within the last 6 months 
• 
HbA1c 6.0 – 10% 
• 
Willingness to enter Part B of the study and be randomised to exercise training or control 
• 
Able to meet the time and physical demands encompassed within the exercise training 
intervention 
 
Exclusion criteria 
• 
Contraindications to magnetic resonance procedures 
• 
Contraindications to exercise training 
• 
Participating in regular purposeful exercise training of vigorous intensity – frequency ≥ 3 sessions per 
week and intensity ≥ 6.0 metabolic equivalents (METs) 
• 
Weight instability or planned/ on-going dietary intervention 
• 
Unable to communicate sufficiently in English 
• 
Co-morbidity that the research team determine to be a contraindication to involvement 
• 
Current smoker 
• 
Uncontrolled hypertension - systolic blood pressure ≥ 160 mmHg and/or diastolic blood pressure ≥ 
100 mmHg 
 
Additional criteria for participants with T2DM/prediabetes 
• 
Taking additional oral anti-diabetic medications to metformin e.g. SGLT2i, GLP-1RA, DPP4 
inhibitors, TZDs within the last 6 months 
• 
Taking insulin 

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11. STUDY PROCEDURES 
 
Verbal eligibility check 
Prior to commencing the study, participants will be contacted via telephone and an initial verbal eligibility 
check will take place (approx. 30 min conversation). Following this, potentially eligible participants will 
be invited to visit the Sir Peter Mansfield Imaging Centre for study assessment visit one. 
Within 72 hours of each visit to a research site (assessment visits and supervised training sessions), 
participants will be contacted via telephone by a member of the research team and a verbal COVID-19 
symptom screening questionnaire will be administered. As per site guidelines, the questionnaire will be 
site-specific to Leicester and Nottingham sites. Furthermore, the COVID-19 symptom screening 
questionnaire will be further completed by participants upon arrival at each study site visit to further 
screen for symptoms.   
 
Assessment visit 1 - Sir Peter Mansfield Imaging Centre (University of Nottingham) 
Informed consent 
Before any study related procedure can take place, the participant must sign and date the most recent 
approved version of the Informed Consent Form. Before consent is obtained the responsible individual 
will ensure that the participant fully understands all aspects of the study. It will also be clearly stated that 
the participant is free to withdraw from the study at any time, for any reason, without prejudice to future 
care, and with no obligation to give the reason for withdrawal. If a participant withdraws from the study 
but does not withdraw their consent, then data already obtained may be used for the study. If a participant 
has withdrawn due to an adverse event, a healthcare professional will follow up as appropriate. 
 
The consent form will be signed and dated based up on an informed decision. The consent process will 
be performed by someone who has received consent training, has been authorised by the Chief 
Investigator, and is named on the delegation of authority log. The original signed form will be retained at 
the study site and copy will be given to the participant. A third copy will be posted to the participant’s GP. 
 
Questionnaires 
Participants will complete several pen and paper based questionnaires to assess their eligibility to 
participate in the study and to provide baseline study data. These will include the following: 
• 
Physical Activity Readiness Questionnaire - to determine participants’ readiness to exercise 
and/or contraindications 
• 
Loughborough University Health Screen Questionnaire – to obtain an overview of participants’ 
medical history, current medications and family history of disease 
• 
Sir Peter Mansfield Imagining Centre Magnetic Resonance Safety Screening Form – to assess 
potential contraindications to MRI

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• 
Demographics & contact details – to determine participants’ date of birth, ethnicity and postal 
address 
 
Anthropometry, blood pressure and finger prick blood test 
Height, body mass and waist circumference will be measured using standard techniques and body mass 
index (BMI) will be calculated. Body fat percentage will be estimated using bioelectrical impedance 
analysis. Blood pressure will be assessed using an automated monitor after participants have sat quietly 
for 10 minutes (average of three measurements). A finger prick blood test will be taken to check 
preliminary eligibility (HbA1c, glucose, lipids) using a point-of-care device. 
 
Magnetic resonance imaging (MRI) 
MRI data will be acquired using a 3T Philips Ingenia MRI scanner. Liver fat quality indices and total 
intrahepatocellular liver fat fractions will be determined using 1H-MRS via the quantification of individual 
lipid group peaks. Liver inflammation, visceral adipose tissue and subcutaneous abdominal adipose 
tissue will be measured via MRI. The techniques used to measure these outcomes have been validated 
and published previously [13,18,19]. 
 
Familiarisation with weighed food records and physical activity monitors 
Participants will be provided with a wrist worn (watch-like) physical activity monitor (GENEactiv) that 
they will wear 24 h/day for the next seven days. Participants will also be shown how to keep an accurate 
weighed food record and will be provided with food scales and diary to facilitate a three-day record within 
the next seven days (two week and one weekend day). 
 
After this session, MRI scans will be assessed to examine whether or not participants have an amount 
of liver fat that if high enough for inclusion in the study (> 5.56%). If liver fat is less than this then 
participants will not be permitted to participate. If liver fat is equal to or greater than this level, then 
participants will be invited to attend study assessment visit two. 
 
Assessment visit 2 - Leicester General Hospital (Leicester Diabetes Centre) or Queens Medical 
Centre Hospital (NIHR Nottingham BRC) 
Fasting blood sample 
Participants will be asked to attend the second study assessment visit having not eaten since the prior 
evening. These visits will therefore occur in the morning (before 10:30). At the start of this visit 
participants will provide a fasting venepuncture blood sample (50 mL blood) after having sat down for 
10 minutes. This sample will be collected from an antecubital vein by an individual trained and 
experienced in phlebotomy. One blood bottle will be sent to hospital pathology labs for the analysis of 
glycated haemoglobin. The remaining samples will be processed immediately and stored until the end 
of the trial within -80oC freezers at the research sites. Following the last participants visit, samples will 

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be transported to Loughborough University (at -80oC) by a commercial courier. Samples will then be 
analysed in batch for: 
 
• 
Lipids 
• 
C-reactive protein 
• 
Liver enzymes 
• 
Glucose 
• 
Insulin 
• 
Nonesterifed fatty acids 
• 
Hepatokines 
• 
Inflammatory proteins 
• 
Genotype (gene variants linked to NAFLD) 
 
Medical evaluation and aerobic fitness 
A healthcare professional will take a full medical history from participants in order to identify any 
factors that may prohibit individuals from completing the study. Blood pressure will be measured, and 
a 12 lead ECG and a physical examination will also be performed. Finally, a symptom limited 
progressive treadmill exercise test will be undertaken on a treadmill. This test will require participants to 
exercise at increasingly harder intensities until volitional exhaustion. A clinical professional will monitor 
participants’ blood pressure and ECG during this test which will be terminated if clinically indicated. 
 
 The physical examination is optional and dependent on findings from Medical History and 12 Lead ECG. It is to be 
performed by individuals qualified to do physical examinations. 
 
 
Randomisation 
After visit two, participants entering Part B of the study will be randomised (1:1) to condition (exercise 
training or control). Randomisation will be performed by the trial statistician (Dr Ghazala Waheed) (see 
section 13 for additional details). The involvement of participants who are only completing Part A of the 
study will end at this point. 
 
Six week intervention 
Participants randomised to control will receive no interventions during the six-week intervention phase. 
During this time, control participants will be asked to not change any aspects of their lifestyles. Ahead 
of week five, participants will be posted a physical activity monitor to wear continuously during week five 
and six. Participants will also complete weighed food diaries during week five (two weekdays and one 
weekend day). 

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Participants randomised to exercise training will complete four exercise training sessions per week 
during the six-week intervention. Each week, at least one session will be supervised by the research 
team whilst the remaining sessions will consist of participants undertaking brisk walking on their own. 
All exercise sessions will be composed of continuous moderate-intensity exercise and last 35 - 50 
minutes in duration (depending on intervention week). The specific details relating to these training 
sessions can be seen in section 12 (Table 2). 
 
Assessment visit 3 (post-intervention) - Sir Peter Mansfield Imaging Centre (University of 
Nottingham) 
The assessments undertaken at visit three will be a repeat of those completed during visit one. 
 
 
Assessment visit 4 (post-intervention) - Leicester General Hospital (Leicester Diabetes Centre) 
or Queens Medical Centre Hospital (NIHR Nottingham BRC) 
The assessments undertaken at visit four will be a repeat of those completed during visit two except that 
a medical examination will not be performed. 

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12. TREATMENT OF TRIAL PARTICIPANTS 
Overview of procedures 
This study will consist of two parts (A and B) and will include two discrete groups of participants. 
1. Obese men with NAFLD – will complete Part A only 
2. Obese men with NAFLD and T2DM/prediabetes – will complete Part A and B 
Table 1 provides an overview of the procedures that participants will undertake as part of this study. 
 
 
Table 1: summary of trial procedures for study participants 
 
Visit 
Duration 
Procedure(s) 
Study Part A – all participants 
Telephone interview 
30 min 
Verbal eligibility check against inclusion / exclusion criteria 
Assessment visit 1 
(SPMIC) 
2 h 
Informed consent, study questionnaires, anthropometry, 
blood pressure, finger prick test, MRI scan 
Assessment visit 2 
(QMC or LGH) 
2 h 
Fasting blood sample, medical examination, peak oxygen 
uptake test 
Study Part B – obese men with NAFLD & 
T2DM/prediabetes only 
3-20 
Exercise training 
(supervised sessions at QMC, 
LGH or Loughborough 
University) 
1 h per 
session 
Heart rate, RPE 
Assessment visit 3 
(SPMIC) 
2 h 
Study questionnaires, anthropometry, blood pressure, MRI 
Assessment visit 4 
(QMC or LGH) 
2 h 
Fasting blood sample, peak oxygen uptake test 
RPE - ratings of perceived exertion, MRI magnetic resonance imaging, HbA1c – glycated haemoglobin, 
SPMIC – Sir Peter Mansfield Imaging Centre, QMC – Queens Medical Centre Hospital, LGH - Leicester 
General Hospital 
 
At the beginning of the study, all potential participants will complete a telephone interview to verbally 
assess eligibility for the study (age, sex, activity status, smoking status, habitual alcohol intake, 

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understanding of English, medical history, medication use, contraindications to the study procedures, 
willingness and ability to undertake six weeks exercise training). Participants who are potentially eligible 
will then be invited to attend the Sir Peter Mansfield Imaging Centre located on the University of 
Nottingham campus for the first study assessment visit. 
 
Prior to each visit to a study site (including assessment visits and supervised training sessions) 
participants will be contacted via telephone by a member of the research team within 72 hours of their 
visit to complete a site-specific COVID-19 symptom screening questionnaire. Upon arrival at the study 
site, participants will verbally complete the COVID-19 symptom screening questionnaire again with a 
member of the research team.  
 
Before participants’ first study visit, they will be asked to consume a normal diet i.e. no unusual feasting 
or energy restriction, in the prior 24 h. No alcohol or high caffeine containing foods will be permitted 
during this time. Participants will also be asked to attend the research centre in the morning having not 
eaten since the prior evening (after 22:00). At this initial visit, a member of the research team will check 
participants’ understanding of the study and written informed consent will be obtained. Participants will 
then complete additional screening and eligibility assessments as detailed in section 
11. Before leaving the research centre, participants will have a physical activity monitor attached to them 
(watch like device) that they will wear for the next seven days. They will also be provided with food 
weighing scales and a food diary to assist them in providing a three-day weighed food record (two week 
and one weekend day) within the subsequent seven days. 
 
After study assessment visit one, the research team will contact participants by telephone to inform them 
whether or not they have sufficiently high liver fat to permit their inclusion in the study. If so, study 
assessment visit two will be arranged. If participants do not meet the inclusion criteria, they will be asked 
to post back the food diary and physical activity monitor using a prepaid envelope already provided. 
These individuals will take no further part in the study. 
 
Participants’ second assessment visit will take place at the Queens Medical Centre Hospital 
(Nottingham) or the Leicester General Hospital (depending on preference). Ahead of this visit, they will 
be asked to consume a ‘normal diet’ in the 24 h beforehand. No alcohol or high caffeine containing foods 
will be permitted during this time. Participants will also be asked to attend the research centre in the 
morning having not eaten since the prior evening (after 22:00). Participants will bring their physical 
activity monitor and food diary with them to this visit. During this visit participants will undertake all of the 
assessments outlined in section 11. The completion of this visit will mark the end of procedures for 
participants who are only completing Part A of the study (obese men with NAFLD). In due course, a  

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member of the study team will contact these individuals via telephone in order to discuss the provision 
of feedback in relation to their results. 
 
Participants progressing to Part B of the study (obese men with NAFLD and T2DM/prediabetes) will 
then be randomised to exercise training or control for six weeks. Participants will be made aware of their 
group assignment via telephone. Control participants will undergo no interventions and will be asked not 
to alter any aspect of their lifestyle during the next six weeks. Ahead of week five, control participants 
will receive a physical activity monitor and food diary in the post to enable them to record their habitual 
physical activity and food intake during weeks five and six. After the intervention phase has ended, 
control participants will complete two additional study assessment visits (visits three and four) to obtain 
post-intervention measurements. These visits will be a repeat of assessment visits one and two 
(excluding the pre-exercise screening in visit two). 
 
Participants randomised to the exercise training group will complete 24 exercise training sessions (35 
to 50 min of moderate-intensity exercise) during the six-week intervention. Four training sessions will be 
completed each week, with at least one of these sessions being supervised by the study team. 
Participants will have the option to undergo these sessions at Loughborough University (National Centre 
of Sport and Exercise Medicine), Nottingham (Queens Medical Centre Hospital) or Leicester (Leicester 
General Hospital) depending on preference. The remainder of the sessions each week to be completed 
by participants will be unsupervised sessions of brisk walking which will be recorded via a heart rate 
monitor and watch. Participants will be supported after each unsupervised session via a telephone call 
from a member of the research team and the intensity and duration of the session will be recorded from 
the watch. Participants in the exercise group will wear a wrist-worn movement sensor throughout the 
entire training phase and will complete weighed food diaries (two week and one weekend day) in the 
fifth and sixth weeks of training. 
 
Exercise training 
The exercise training program in Part B of this study is based upon joint guidelines published by the 
European Association for the Study of Liver Disease (EASL), European Association for the Study of 
Diabetes (EASD) and European Association for the Study of Obesity (EASO); which state that 
individuals with NAFLD should aim to complete 150 to 200 min per week of exercise [20]. Participants 
will therefore complete four, 50 min exercise training sessions each week. Each training session will be 
composed of moderate-intensity continuous exercise at a target intensity that will be determined using 
a combination of age-predicted maximum heart rate (70-75%) and RPE (13-14). 
 
During each week of exercise training, participants will complete four exercise training sessions, of which at 
least one of these sessions will be a supervised training sessions. The duration of exercise training will 
progress from week one to week six to support participants’ completion of the programme (Table 2). 

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Supervised exercise training sessions will include a 10-minute warm-up and five min cool down at a self-
selected light intensity (RPE 11 – ‘fairly light’). All supervised training sessions will take place on a 
treadmill (walking or jogging); however, a cycle ergometer will be available for participants to use should 
they feel discomfort with prolonged upright exercise. Supervised training sessions will occur Monday to 
Friday. 
 
Participants will undertake the non-supervised training sessions in their habitual environment e.g. local 
park. In these sessions, participants will undertake brisk walking at an intensity corresponding to 70- 
75% of maximum age-predicted heart rate and/or an RPE of 13-14. The intensity and duration of 
sessions will be monitored using a heart rate monitor and accompanying watch. 
 
Table 2: overview of the supervised & unsupervised moderate-intensity continuous exercise 
training sessions 
Supervised exercise sessions 
Unsupervised 
exercise sessions 
Training 
Week 
Warm up 
duration 
(min) 
Cool down 
duration 
(min) 
Main body 
duration 
(min) 
Brisk walking 
session duration 
(min) 
Total weekly 
exercise 
duration (min) 
1 
10 
5 
20 
45 
150 
2 
10 
5 
25 
45 
165 
3 
10 
5 
30 
45 
180 
4 
10 
5 
30 
50 
185 
5 
10 
5 
35 
50 
200 
6 
10 
5 
35 
50 
200 
NB: four exercise training sessions will be completed each week, of which at least one session will be a 
supervised exercise training session and the remainder will be an unsupervised session. 
 
Given funding limitations, control participants will not be offered the exercise intervention at the end of 
the study. Instead, the research team will offer to provide support to participants wishing to become 
more active beginning with a discussion of their physical activity and fitness data obtained from the 
study. 
 
Remuneration and expenses 
Participants will not be paid for their participation in Part A or B of this study. Travel expenses will be 
provided for participants 

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13. SAFETY REPORTING  
Definitions 
Adverse Event (AE) 
An AE or adverse experience is: 
Any untoward medical occurrence in a patient or clinical investigation participants, which does not 
necessarily have to have a causal relationship with this treatment. 
An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory 
finding), symptom or disease temporally associated with the study, whether or not considered related to 
the study. 
 
Adverse Reaction (AR) 
All untoward and unintended responses related to the study. 
All cases judged by either the reporting medically qualified professional or the sponsor as having a 
reasonable suspected causal relationship to the study qualify as adverse reactions. There are no 
expected ARs in this study. 
 
Severe Adverse Events 
To ensure no confusion or misunderstanding of the difference between the terms "serious" and "severe", 
which are not synonymous, the following note of clarification is provided: 
The term "severe" is often used to describe the intensity (severity) of a specific event (as in mild, 
moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical 
significance (such as severe headache). This is not the same as "serious," which is based on 
patient/event outcome or action criteria usually associated with events that pose a threat to a 
participant's life or functioning. Seriousness (not severity) serves as a guide for defining regulatory 
reporting obligations. 
 
Serious Adverse Event or Serious Adverse Reaction 
A serious adverse event or reaction is any untoward medical occurrence that at any dose: 
• 
Results in death, 
• 
Is life-threatening, 
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. 
• 
Requires inpatient hospitalisation or prolongation of existing hospitalisation, 
• 
Results in persistent or significant disability/incapacity, or 

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• 
Is a congenital anomaly/birth defect. 
• 
Other important medical events* 
*Other events that may not result in death, are not life threatening, or do not require hospitalisation, may be 
considered a serious adverse event when, based upon appropriate medical judgement, the event may 
jeopardise the patient and may require medical or surgical intervention to prevent one of the outcomes listed 
above. 
 
Expected Serious Adverse Events/Reactions 
This study is a non-invasive lifestyle related study and therefore no SAE/Rs are expected. 
 
 
Suspected Unexpected Serious Adverse Reactions 
This study is a non-invasive lifestyle related study and therefore no SUSARs are expected. 
 
 
Reporting Procedures for All Adverse Events 
All AEs occurring during the study observed by investigators or reported by the participant, whether or 
not attributed to study, will be recorded on the CRF. The following information will be recorded: 
description, date of onset and end date, severity, assessment of relatedness to study, other suspect 
device and action taken. Follow-up information should be provided as necessary. 
 
AEs considered related to the study as judged by a medically qualified investigator or the sponsor will 
be followed until resolution or the event is considered stable. All related AEs that result in a participant’s 
withdrawal from the study or are present at the end of the study, should be followed up until a satisfactory 
resolution occurs. 
 
Reporting Procedures for Serious Adverse Events 
All SAEs must be reported to the Sponsor within one working day of discovery or notification of the 
event. The Sponsor will perform an initial check of the information and ensure that it is reviewed at the 
next R&D Management meeting. All SAE information must be recorded on an SAE form and sent to the 
Sponsor using the appropriate reporting form and the contact details on there. Additional information 
received for a case (follow-up or corrections to the original case) needs to be detailed on a new SAE 
form which must be sent to the Sponsor using the appropriate reporting form and the contact details on 
there. 
 
The Sponsor will report all SUSARs to the Research Ethics Committee concerned. Fatal or life- 
threatening SUSARs must be reported within 7 days and all other SUSARs within 15 days. The Chief 

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Investigator will inform all investigators concerned of relevant information about SUSARs that could 
adversely affect the safety of participants. 
 
In addition to the expedited reporting above, the Chief Investigator shall submit once a year throughout 
the study or on request an Annual Report to the Ethics Committee which lists all SAEs / SUSARs that 
have occurred during the preceding 12 months. 

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14. STATISTICS 
Description of statistical methods 
Descriptive statistics will be calculated to outline the characteristics of the study sample. Normality of data 
will be assessed using histograms and box plots, while further analysis of skewness and kurtosis will be 
conducted if normality is not clear from the histograms and box plots. Depending on the distribution of data, 
participant characteristics will be reported as mean (SD) or median (IQR) and number (percentage) for 
continuous variables and for categorical variables respectively. 
 
For part A of the study, data will be checked for parametric assumptions. Independent samples t-tests 
will used to analyse differences in continuous variables between study groups (obese men with NAFLD 
and obese men with NAFLD and T2DM in part A of the study). To examine factors associated with NAFLD 
and T2DM, univariate logistic regression models will be performed with NAFLD and T2DM (diabetic 
individuals with NAFLD vs. non-diabetic individuals with NAFLD) as the dependent variable. All variables 
significantly associated with NAFLD and T2DM in univariate logistic regression analyses will be included 
in multivariate logistic regression analysis. 
 
For part B of the study, baseline descriptive statistics will be summarised by treatment arm (Intervention 
vs control). Data will be checked for parametric assumptions. For the primary outcome, change in hepatic 
saturated lipid index (%) from baseline to six weeks due to moderate-intensity exercise training in men 
with NAFLD and T2DM/prediabetes, treatment arms will be compared using linear regression modelling. 
The analysis will include a binary indicator for randomisation group as the explanatory variable, terms for 
the stratification category (ethnicity) and adjustments for the baseline measure of the outcome (hepatic 
saturated lipid index). 
 
Complete case analysis will be utilised for the primary analysis. Intention to treat will be carried out as 
sensitivity analysis, using multiple imputation for missing values. Secondary outcomes will be analysed 
using similar methods as the main analysis, with an appropriate model selected dependent on the 
distribution of the outcome. 
 
The assumptions associated with each model will be assessed and where these are not met alternative 
models or parameterisations will be considered. A value of P<0.05 will be considered statistically 
significant for all analyses. Statistical analyses of the baseline data and all future analysis will be carried 
out using STATA version 15. 

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The Number of Participants 
The sample size for the research outlined in this protocol was undertaken by a statistician in the Leicester 
Clinical Trials Unit. The sample size calculations were informed by data published in a previous study which 
validated the method for measuring hepatic fat quality via 1H-MRS[16]. 
 
Part A of this research (cross-sectional analyses) was powered (80%) to detect a difference in the liver 
saturated fat indices of 0.5 between men with NAFLD with T2DM versus non-diabetic men with NAFLD 
(alpha 5%, SD 0.4 in each group). 28 participants are needed to provide adequate statistical power for this 
analysis (14 in each group). Based on the same data, the power calculation showed that 24 participants are 
needed for Part B of the outlined research (RCT). This is inflated to 26 to allow for expected drop-out (10%). 
 
Please note that the baseline data for all participants entering Part B of this research will be used in Part A 
and therefore the cross-sectional analyses will include data from 26 men with NAFLD & T2DM and 14 non- 
diabetic men with NAFLD. 
 
Procedure for dealing with missing and spurious data 
Missing data will be replaced using multiple imputation or another appropriate method. In Part B, 
participants completing less than 85% of all training sessions will not be included in the final analysis. 

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15. DIRECT ACCESS TO SOURCE DATA / DOCUMENTS 
Beyond the study team, 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. 

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16. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
Loughborough University as sponsor operate a risk-based audit programme to which this study will be 
subject. The research team will be responsible for all elements of study management on an on-going 
basis. A documented monitoring log and audit trail will be maintained throughout the lifetime of the study. 
The CI and PIs will oversee the set-up and conduct of study procedures at each site. All source data 
and study documents will be made available for Sponsor monitoring, and any external audits and 
inspections as appropriate, for example by the Research Ethics Committee. 
 
It is acknowledged that accidental protocol deviations can happen at any time; however, thorough 
training of research staff and appropriate utilisation of SOPs will limit the likelihood of deviations 
occurring. One-off protocol deviations will be documented on the relevant forms and the Chief 
Investigator will be notified. Frequent protocol deviations will be escalated to the TSC and appropriate 
actions considered. 

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17. CODE OF PRACTICE AND REGULATIONS 
 
 
Ethics 
Approval from Loughborough University (sponsor), a Local Research Ethics Committee (REC), the 
Health Research Authority (HRA), University of Nottingham, University Hospitals of Leicester NHS Trust 
R&D and Nottingham University Hospitals Trust R & D will be sought prior to the commencement of the 
research. This will ensure that all ethical and indemnity issues are dealt with appropriately. The research 
protocol, Participant Invitation Letter, Informed Consent Form, Participant Information Sheet and any 
proposed advertising material will be submitted to the sponsor, main REC, and host institutions for 
approval. All formal correspondence with NHS REC and host institutions will be retained. The Chief 
Investigator will submit and, where necessary, obtain approval from the above parties for all substantial 
amendments to the original approved documents. Substantial amendments that require review by NHS 
REC will not be implemented until that review is in place and other mechanisms are in place to implement 
at site. The Chief Investigator will ensure that this study is conducted in full conformity with relevant 
regulations and with the International Conference on Harmonisation Guidelines for Good Clinical 
Practice (IHC-GCP). 
 
Participants will be free to withdraw at any time from the study without giving a reason and without their 
legal rights being affected. We do not anticipate any harm, discomfort or risk to any participant enrolled 
in this study. The overall care and comfort of the participant will be considered paramount at all times 
during the study. 
 
Sponsor Standard Operating Procedures 
All relevant SOPs will be followed to ensure that this study complies with all relevant legislation and 
guidelines. 
 
Declaration of Helsinki 
The Investigator will ensure that this study is conducted in full conformity with the current revision of the 
Declaration of Helsinki (last amended October 2000, with additional footnotes added 2002 and 2004). 
 
ICH Guidelines for Good Clinical Practice 
The Investigator will ensure that this study is conducted in full conformity with relevant regulations and 
with the ICH Guidelines for Good Clinical Practice (CPMP/ICH/135/95) July 1996. 
 
Approvals 
Once Sponsor authorisation has been confirmed, the protocol, Informed Consent Form, Participant 

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Information Letter, Participant Information Sheet, GP correspondence documents and any proposed 
advertising material will be submitted to an appropriate Research Ethics Committee (REC), regulatory 
authorities (HRA), and the host institution for written approval. 
 
Once Sponsor authorisation has been confirmed, the Chief Investigator will submit and, where necessary, 
obtain approval from the above parties for all substantial amendments to the original approved 
documents. 
 
The Chief Investigator will formally notify NHS REC and host institutions of study end once the trial is 
closed. 
 
Participant Confidentiality 
The trial staff will ensure that the participants’ anonymity is maintained. Participants will be identified only 
by initials and a participant’s ID number on the CRF and any electronic database. All documents will be 
stored securely and only accessible by trial staff and authorised personnel. Direct access to all documents 
will be granted where appropriate to authorised representatives from the sponsor, host institution and the 
regulatory authorities for monitoring, audits and inspections. The study will comply with the Data 
Protection Act which requires data to be anonymised as soon as it is practical to do so. 
 
Other Ethical Considerations 
In this study, it is possible that the study team may identify various health issues during the process of 
screening and trial assessments. As examples, magnetic resonance imaging may identify liver masses, 
or ECG traces may identify abnormal heart electrical rhythms. In these instances, the study team will 
immediately inform the participant and provide written notification. If the participant consents, this 
information will also be shared with their GP so that appropriate follow-up can occur. 

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18. DATA HANDLING AND RECORD KEEPING 
All data collected will be kept strictly confidential and in accordance with the Data Protection Act 1998. 
The research staff will ensure that the participants’ anonymity is maintained. On all study-specific 
documents, other than the signed consent form and enrolment log, the participant will be identified by 
initials and/or a participant ID number, not by name. 
 
All research data will be kept in a secure location accessible only by named members of the research 
team during the active phase of the study and until the data have been analysed. It will then be archived 
for five years after the end of the trial and subsequently destroyed. 
 
Direct access to information gathered in this study will only be available to individuals who have been 
granted access. The sponsor, host institution and regulatory authorities can permit trial related 
monitoring, audits and inspections. 
 
All study documentation containing identifiable patient data will be managed in accordance with ICH- 
GCP, Research Governance Framework for Health and Social Care and the Data Protection Act. 
Information will only be obtained from the patient if necessary for the study. 
 
All electronic data will be stored on secure university (Loughborough University) or hospital (NUH / UHL) 
network drives, to which only the relevant study staff have access, which is granted by the Chief 
Investigator. All study documents and data will be kept for 5 years. Paper copies of the CRFs will be 
stored in locked cabinets at research sites. 

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19. STUDY GOVERNANCE 
 
 
PPI Involvement Group: the NIHR Leicester BRC has an established diabetes PPI group who 
frequently provide support for research projects led by researchers within the Diabetes Research Centre 
(University of Leicester) and Loughborough University (School of Sport, Exercise and Health Sciences). 
Members of this group reviewed the research outlined in this protocol before the project underwent peer 
review by Diabetes UK and have supported the development of study materials for this proposal (e.g. 
Participant Information Sheet, Informed Consent Form, Case Report Forms). Two individuals from this 
group will become members of the TSC to help facilitate the smooth and timely conduct of the research. 
 
Trial steering committee (TSC) 
A TSC will provide oversight of this study with meetings convened every other month in the first 12 
months of the study and quarterly thereafter. Membership of this committee will comprise the CI, PIs, 
Co-investigators and PPI representatives. 
 
Data safety monitoring committee 
There will be no data safety monitoring committee for this study. All data safety matters will be dealt with 
immediately by the Chief Investigator and reviewed by the study team at TSCmeetings. 
 
Access to the final study dataset 
The Chief Investigator will hold the master copy of the final study dataset. All co-investigators may be 
permitted access to this file as deemed necessary by the Chief Investigator. 

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20. FINANCE AND INSURANCE 
This research will be funded by Diabetes UK (Early Career Small Grant – Dr James King) and the NIHR 
Nottingham and Leicester BRCs. All costs due to be incurred within this research project are detailed 
below. Please note that this study will additionally be supported by the established infrastructure within 
the NIHR Leicester and Nottingham BRCs (costs not identified here). Support will include a Post- 
Doctoral Research Associate (0.5 WTE), PhD student and Administrator who will form the basis of the 
study team. Medical support for clinical procedures will also be provided by clinical research fellows and 
cardiac nurses employed within the BRCs. 
 
Table 2: itemised research study costings 
 
Cost of procedure 
Funder 
Imaging procedures 
£16,500 
Diabetes UK - £13,000 
Nottm BRC - £3,500 
HbA1c (hospital labs) 
£480 
Nottm BRC - £480 
In-house blood analysis 
£5,712 
Diabetes UK - £1,500 
Leicester BRC - £4,212 
Participant travel 
£1,062 (est. maximum amount) 
Leicester BRC - £1,062 
Investigator travel 
£1,170 (est. maximum amount) 
Diabetes UK - £500 
Leicester BRC - £670 
Total cost 
£24,924 
 
 
Sponsorship for this study will be provided by Loughborough University. Loughborough University will 
therefore provide indemnity to meet the potential legal liabilities of the sponsor for harm to participants 
arising from the management of the research; and for harm to participants arising from the design of the 
research. If a patient is harmed due to negligence this would be covered by the NHS Trust(s) indemnity 
arrangements for all participants in clinical trials. If a study participant wishes to make a complaint about 
any aspects of the way they have been treated or approached during the research project, the standard 
National Health Service complaint system will be available to them 

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21. PUBLICATION / DISSEMINATION POLICY 
At the end of this study the data will be tabulated and analysed statistically. These data will then be 
written up within a final report that will be submitted by the Chief Investigator to the primary study sponsor 
(Diabetes UK) within six months of study completion. Within one year after the end of the study, the Chief 
Investigator will also submit a final report to REC outlining the results and any publications/abstracts. All 
investigators identified on this document will be acknowledged on the final report to Diabetes UK and 
REC. It will be made clear in the PIS that this final report will subsequently be made available to 
participants after the study and individual feedback will be available in the desired format e.g. verbally or 
in writing. 
 
It is envisaged that results from this study will be published in relevant peer-reviewed scientific journals 
and will also be submitted for conference presentations. The TSC will oversee the development of 
manuscripts and abstracts; and the list / order of authors shall be decided at the point of submission. 
The guidelines outlined by the scientific journal in question shall be used to determine the criteria for 
authorship for each manuscript on a case-by-case basis. In all presentations and publications, all 
funders will be appropriately acknowledged (Diabetes UK, NIHR Nottingham and Leicester Biomedical 
Research Centres). 

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22. REFERENCES 
 
1. Buzzetti E, Pinzani M, Tsochatzis EA (2016) The multiple-hit pathogenesis of non-alcoholic 
fatty liver disease (NAFLD). Metabolism. 65(8): 1038-1048. 
 
2. Younossi ZM, Blissett D, Blissett R, Henry L, Stepanova M, Younossi Y, Racila A, Hunt S, 
Beckerman R (2016) The economic and clinical burden of nonalcoholic fatty liver disease in 
the United States and Europe. Hepatology. 64(5): 1577-1586. 
 
3. Byrne CD and Targher G (2015) NAFLD: a multisystem disease. J Hepatol. 62(s1): s47-s64. 
 
4. Bril F, Barb D, Portillo-Sanchez P, Biernacki D, Lomonaco R, Suman A, Weber MH, Budd JT, 
Lupi ME, Cusi K (2016) Metabolic and histological implications of intrahepatic triglyceride 
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5. Korenblat KM, Fabbrini E, Mohammed BS, Klein S (2008) Liver, muscle, and adipose tissue 
insulin action is directly related to intrahepatic triglyceride content in obese subjects. 
Gastroenterology. 134(5): 1369-1375. 
 
6. Cusi K (2016) Treatment of patients with type 2 diabetes and non-alcoholic fatty liver disease: 
current approaches and future directions. Diabetologia. 59(6): 1112-1120. 
 
7. Mishra A and Younossi ZM (2012) Epidemiology and natural history of non-alcoholic fatty liver 
disease. Journal of clinical experimental hepatology. 2(2): 135-144. 
 
8. Targher G, Byrne CD, Lonardo A, Zoppini G, Barbui C (2016) Non-alcoholic fatty liver disease 
and risk incident cardiovascular disease: a meta-analysis. Journal of Hepatology. 65(3): 589- 
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9. Luukkonen PK, Zhou Y, Sadevirta S, Leivonen M, Arola J, Oresic M, Hyotylainen T, Yki- 
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George J (2008) Non-invasive assessment of hepatic lipid composition: advancing 
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14. Haus JM (2013) Improved hepatic lipid composition following short-term exercise in 
nonalcoholic fatty liver disease. Journal of clinical endocrinology and metabolism. 98 (7): 
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15. Johnson NA et al (2009) Aerobic exercise training reduces hepatic and visceral lipids in obese 
individuals without weight loss. Hepatology. 50(4): 1105-1112. 
 
16. Sullivan S, Kirk EP, Mittendorfer B, Patterson BW, Klein S. Randomised trial of exercise effect 
on intrahepatic triglyceride content and lipid kinetics in nonalcoholic fatty liver disease. 
Hepatology. 55(6): 1738-1745. 
 
17. Szczepaniak LS, Nurenberg P, Leonard D, Browning JD, Reingold JS, Grundy S, Hobbs HH, 
Dobbins RL. Magnetic resonace spectroscopy to measure hepatic triglyceride content: 
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Endocrinolgy and Metabolism. 288(2): E462-468. 
 
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low glycaemic index food: a randomised crossover study. Diabetes, Obesity and Metabolism. 
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fatty liver disease. Journal of Hepatology. 64(6): 1388-1402.