The Effect of Acute, Passive Heating on Glucose Tolerance in Individuals With Type 2 Diabetes Mellitus: a Randomised, Balanced Crossover, Control Trial
Phase: NA
Conditions: Diabetes Mellitus, Type 2
Sponsor: University of Portsmouth

 
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Study Title:  To bathe, or not to bathe? The effect of acute, passive heating on 
glucose tolerance in individuals with type 2 diabetes mellitus: a randomised, 
balanced crossover, control trial. 
 
NCT03798717 
Ethics Ref: 18/SC/0581 
Date and Version No: 1.2 05-04-2019  
              
Chief Investigator: 
Dr. Anthony Shepherd  
 
Investigators:  
See protocol development team below  
Sponsor:  
Portsmouth Hospitals NHS Trust 
Funder: 
University of Portsmouth  
Signatures:  
 
 
Protocol Development Team: 
Chief Investigator  
 
- Dr Anthony Shepherd 
(University of Portsmouth) 
Principal Investigator         
- Mr Thomas James               (University of Portsmouth) 
Co- investigator 1 
 
- Dr Jo Corbett  
 
(University of Portsmouth) 
Co- investigator 2 
 
- Dr Joseph Costello              (University of Portsmouth) 
Co- investigator 3                   - Prof. Mike Tipton                 (University of Portsmouth) 
Co- investigator 4                   - Prof. Janis Shute                 (University of Portsmouth) 
Co- investigator 5                   - Dr Zoe Saynor                     (University of Portsmouth)  
Co- investigator 6                   - Dr. Iain Cranston                 (Queen Alexandra Hospital) 
Co- investigator 7                   - Prof. Michael Cummings     (Queen Alexandra Hospital) 
Co- investigator 8 
 
- Mr Connor Morgan   
(University of Portsmouth) 
Co- investigator 9 
 
- Mr Billy Hopkins 
 
(University of Portsmouth) 
Co- investigator 10 
 
- Miss Emily Windsor   
(University of Portsmouth) 
Co-investigator 11                  -Ms Sharon Allard                  (Queen Alexandra Hospital) 
 

 
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TABLE OF CONTENTS 
 
1. 
AMENDMENT HISTORY 
6 
2. 
SYNOPSIS 
7 
3. 
ABBREVIATIONS 
8 
4. 
BACKGROUND AND RATIONALE 
9 
5. 
PRELIMINARY STUDIES AND EXPERIENCE OF INVESTIGATORS 
9 
6. 
AIMS AND OBJECTIVES 
9 
6.1 
Primary Objective 
9 
6.2 
Secondary Objectives 
9 
7. 
STUDY DESIGN 
10 
7.1 
Summary of Study Design 
10 
7.2 
Primary and Secondary Endpoints/Outcome Measures 
10 
8. 
STUDY PARTICIPANTS 
10 
8.1 
Study Setting 
10 
8.2 
Overall Description of Study Participants 
10 
8.3 
Eligibility Criteria 
10 
9. 
SAMPLING 
11 
10. 
STUDY PROCEDURES 
11 
10.1    Recruitment 
11 
10.2   Screening and Enrolment 
11 
10.3 Randomisation 
11 
10.4 Study Assessments 
12 
10.5 
Discontinuation/Withdrawal of Participants from Study Treatment 
12 
10.6 
Definition of End of Study 
12 
11. 
INTERVENTIONS 
13 
11.1 
Description of Study Intervention / Treatment 
13 
11.2     Adherence to Study Treatment 
13 
11.3     Accountability of the Study Treatment 
13 
11.4     Concomitant Medication / Therapies 
13 
12. 
ASSESSMENT OF SAFETY (if applicable) 
13 

 
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12.1 
Definitions 
13 
12.2 
Reporting Procedures for Serious Adverse Events 
13 
12.3 
Recording and Reporting Procedures for All Adverse Events 
14 
13. 
Data Handling and Record Keeping 
14 
13.1 
Data Collection Forms 
14 
13.2     Data Management 
14 
14. 
DATA ANALYSIS 
14 
14.1      Description of Analysis Populations 
14 
14.2      Analysis of Endpoints 
14 
14.3     Procedure for Dealing with Missing, Unused and Spurious Data 
14 
14.4     Procedures for Reporting any Deviation(s) from the Original Statistical Analysis Plan
 
14 
14.5      Interim analysis and criteria for early study termination 
15 
15. 
ETHICS 
15 
15.1 
Participant Confidentiality 
15 
15.2     Other Ethical Considerations 
15 
15.3     Declaration of Helsinki 
15 
15.4     ICH Guidelines for Good Clinical Practice 
15 
16. 
PATIENT PUBLIC INVOLVEMENT (PPI) 
15 
16.1    Study design 
16 
16.2    Study implementation 
16 
16.3    Dissemination 
16 
17. 
FINANCING AND INSURANCE 
16 
17.1    Research Costs 
16 
17.2    Service Support Costs 
16 
17.3    Excess Treatment Costs 
16 
17.4    Study Sponsorship 
16 
18. 
TIMETABLE AND ORGANISATIONAL CHART 
16 
19. 
RESOURCES, EQUIPMENT AND PHYSICAL FACILITIES 
17 
20. 
DISSEMINATION AND OUTCOME 
17 
21. 
REFERENCES 
17 
22. 
APPENDIX 1     SCHEDULE OF PROCEDURES 
17 

 
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23. 
APPENDIX 2    STUDY FLOW CHART 
17 
24. 
APPENDIX 3    PARTICIPANT INFORMATION SHEET 
17 
25. 
APPENDIX 4    INFORMED CONSENT FORM 
17 
26. 
APPENDIX 5    SAMPLE QUESTIONNAIRES 
17 
27. 
APPENDIX 6    SAMPLE DATA COLLECTION FORMS 
17 
28. 
APPENDIX 
7 
 
 
 
DRUG 
INFORMATION 
(SUMMARY 
OF 
PRODUCT 
CHARACTERISTICS) 
17 
29. 
APPENDIX 8 MANUFACTURERS BROCHURE FOR NOVEL EQUIPMENT 
17 
30. 
APPENDIX 9 CONTRACTUAL AGREEMENTS WITH OUTSIDE CONSULTANTS / 
COLLABORATORS / INSTITUTIONS (E.G. INDUSTRY, CONTRACT RESEARCH 
ORGANISATIONS) 
17 
 
 
 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Author(s) of changes 
Details of Changes 
made 
1 
1 
n.a 
Dr Shepherd  
Changes requested by 
REC. Please see letter 
in site file.  
2 
1.2 
 
Dr Shepherd 
Added research nurse 
and 
recruitment 
via 
primary care.  
1. 
AMENDMENT HISTORY 

 
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2. 
SYNOPSIS 
 
Study Title 
To bathe, or not to bathe. The effect of acute, passive heating on 
glucose tolerance in individuals with type 2 diabetes mellitus: a 
randomised, balanced crossover, control trial. 
Internal ref. no. 
N/A 
Problem statement 
Type 2 diabetes mellitus (T2DM) is characterised by chronic high blood 
sugar concentration (hyperglycaemia) and insulin resistance leading to a 
reduction in insulin sensitivity. These hyperglycaemic excursions can 
seriously impact metabolic, micro and macrovascular health. The total 
cost of the direct and indirect care of individuals with diabetes (~90% 
T2DM) in the UK is £23.7 billion, equating to ~20% of the annual national 
health service (NHS) budget, with this projected to become unsustainable. 
Low-cost interventions to improve glycaemic control in these individuals 
are therefore warranted. Current interventions include pharmaceuticals, 
exercise and calorie restrictive diets. Pharmaceutical interventions carry 
a high financial cost, while exercise and diet programmes have a low 
adherence rate in individuals with T2DM. 
Heat therapy offers one potential low cost therapy. Immersion in a hot tub 
for 30 mins.day-1 for 10 days has been shown to reduce fasting plasma 
[glucose] and HbA1c in individuals with T2DM, which may be explained 
by acute (e.g. muscle) and chronic (e.g. reduced inflammatory status and 
increased heat shock proteins (HSP)) adaptations, although experimental 
evidence for these hypothesis are sparse. Other potential benefits include 
improved glycaemic control, insulin sensitivity, elevated resting metabolic 
rate and improved micro- and macrovascular function.  
The aim of the present study is to determine whether acute hot water 
immersion can improve glucose tolerance in individuals with T2DM and 
whether it is more beneficial to undertake this before or after a OGTT. 
Research question / 
hypothesis 
In individuals with type 2 diabetes mellitus:   
 
Primary question: 
1. Does an acute bout of passive, warm water therapy reduce 
plasma [glucose]?  
 
Secondary questions:  
1. Does plasma [glucose] reduce more if the passive, warm water 
therapy is conducted before or after the OGTT?  
2. Does plasma [insulin] reduce more if the passive, warm water 
therapy is conducted before or after the OGTT?  

 
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3. Does insulin sensitivity increase following an acute bout of warm 
water therapy? 
4. Does fuel utilisation alter during and following an acute bout of 
warm water therapy?  
5. Does cardiovascular status change during or after an acute bout 
of warm water therapy?  
6. Does eHSP increases during and following an acute bout of warm 
water therapy? 
7. Does inflammatory status change during or after an acute bout of 
warm water therapy?  
Study Design 
Randomised, balanced crossover trial, with 3 conditions:  
 
1) OGTT in thermoneutral air (~ 23°C air),  
 
2) Passive heating after an OGTT  
 
and  
 
3) Passive heating before OGTT  
 
For conditions 2 and 3, Trec will be clamped at ~38.5°C using warm 
water. 
Study Participants 
Adults ≥ 35 years, diagnosed with T2DM as defined by the world health 
organisation (WHO)  
Planned Sample Size 
We aim to recruit 20 individuals with T2DM. 
Follow-up duration 
7 days 
Planned Study Period 
18 months 
Primary Objective 
To determine whether an acute bout of passive, warm water therapy 
reduces area under the curve (AUC) [glucose] in individuals with T2DM 
during an OGTT? 
Secondary Objectives In individuals with type 2 diabetes mellitus:   
 
1. Does plasma [glucose] reduce more if the passive, warm water 
therapy is conducted before or after the OGTT?  
2. Does plasma [insulin] reduce more if the passive, warm water 
therapy is conducted before or after the OGTT?  
3. Does insulin sensitivity increase following an acute bout of warm 
water therapy? 
4. Does fuel utilisation alter during and following an acute bout of 
warm water therapy?  
5. Is cardiovascular status altered during or after an acute bout of 
warm water therapy?  

 
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6. Does eHSP increases during an acute bout of warm water 
therapy? 
7. Does inflammatory status reduce during or after an acute bout of 
warm water therapy? 
Primary Endpoint 
To determine whether an acute bout of passive, warm water therapy 
reduces area under the curve (AUC) [glucose] in individuals with T2DM 
during an OGTT? 
Secondary Endpoints 
All other endpoints, including: plasma [insulin], insulin sensitivity, 
substrate oxidation, [eHSP70], inflammatory markers and cardio vascular 
measures (e.g. heart rate, blood pressure). 
Intervention (s) 
Acute (60 min) bouts of warm water therapy aiming to clamp Trec at 
38.5°C (~39°C water temperature, ~ 90%RH)..  

 
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3. 
ABBREVIATIONS 
AE 
 
Adverse event 
ANOVA  
Analysis of variance 
AUC   
Area under the curve 
CF  
 
Cystic fibrosis 
CGM   
Continuous glucose monitoring 
CPET   
Cardiopulmonary exercise test 
CRF   
Clinical records file 
DOVE   
Dysglycaemia, Oxidative stress and the Vascular Endothelium 
ECG   
Electrocardiogram  
EDTA   
Ethylenediaminetetraacetic acid 
eGFR   
Estimated glomerular filtration rate 
eHSP   
Extracellular heat shock protein 
GCP 
 
Good clinical practise 
Hb  
 
Haemoglobin 
HbA1c   
Haemoglobin adult type 1c (Glycated haemoglobin) 
Hct  
 
Haematocrit 
HEC   
Hyperinsulinaemic-euglycaemic clap 
HIIT  
 
High intensity interval training 
HR  
 
Heart rate 
HRV   
Heart rate variability 
HSP   
Heat shock protein 
iHSP   
Intracellular heat shock protein 
IL 
 
Interleukin 
INM  
 
Institute of naval medicine 
LF  
 
Low frequency 
NHS 
 
National Health Service 
NIHR   
National institute for health research 
NIRS  
Near infrared spectroscopy 
NO  
 
Nitric oxide 
NRES   
National research ethics service 

 
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OGIS   
oral glucose insulin sensitivity 
OGTT  
Oral glucose tolerance test 
PCPI   
Patient, carer and public involvement 
PPI  
 
Patient public involvement 
QUICKI         Quantitative insulin sensitivity check index 
REC   
Research ethics committee 
RMR   
Resting metabolic rate 
RMSSD  
Root mean square of the successive differences 
RSA   
Respiratory sinus arrhythmia  
SAE 
 
Serious adverse event 
T2DM  
Type 2 diabetes mellitus 
TNF-α   
Tumour necrosis factor alpha 
Trec  
 
Rectal temperature 
UoP   
University of Portsmouth 
WHO   
World health organisation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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4. 
BACKGROUND AND RATIONALE 
Current estimates suggest 422 million people worldwide live with a form of diabetes, of which 
~ 90% have T2DM [WHO, 2016]. The total cost of the direct and indirect care of individuals 
with diabetes in the UK is £23.7 billion, equating to ~ 20% of the annual NHS budget [Hex et 
al., 2012], this figure is expected to rise to £39.8 billion by the year 2035 [Hex et al., 2012; 
Shaw et al., 2010]. Approximately 85-90% of cases of T2DM arise from a poor lifestyle and 
obesity [ERFC, 2010; George et al., 2013; Kelly et al., 2008], with the other 10-15% resulting 
from genetic predispositions [Daousi et al., 2006; DPPRG, 2002]. Current interventions include 
pharmaceutical treatments, exercise and calorie restrictive diets, which aim to improve 
glycaemic control. However, pharmaceutical interventions carry a high financial cost [Cheng 
and Fantus, 2005; Nathan et al., 2006], while exercise and diet programmes have a low 
adherence rate in individuals with T2DM [Ary et al., 1986; Barnard et al., 2000; Clark, 1997; 
Glasgow et al., 1997; Nathan et al., 2006; Stunkard and Messick, 1985]. With the population 
of individuals with T2DM continuing to increase, the costs associated with the clinical care of 
these individuals are likely to become unsustainable. Simple, inexpensive interventions to 
improve the clinical profile of this group are therefore needed.   
 
One such strategy could be passive heating. Preliminary evidence suggests passive heating 
may have beneficial effects for metabolic health in animal models [Bathaie et al., 2010; Chung 
et al., 2008; Gupte et al., 2009; Gupte et al., 2010; Kokura et al., 2007; Morino et al., 2008; ] 
and in humans [Chung et al., 2008; Faulkner et al., 2017; Hooper, 1999; Rivas et al., 2016; ]. 
This review will focus on the use of warm water and air, and proposed mechanisms of actions 
for these therapeutic aids in individuals with T2DM.   
 
In 1999, the use of hot tubs (38-41°C , 30 mins / day for 3 weeks) was shown to reduce fasting 
plasma glucose concentrations by ~ 14% (1.3 mmol.L-1) and decrease HbA1c by ~10-11 
mmol/mol in 8 adults with T2DM [Hooper, 1999]. Given the rate of turnover in haemoglobin 
this reduction is surprising as the treatment period was run over 3 weeks and the total 
haemoglobin turnover takes ~115 days [Cohen et al., 2008]. Rivas et al., (2016) is the only 
other study to date that has investigated how passive heating effects glycaemic control in 
individuals with T2DM since Hooper (1999). In contrast, Rivas et al. (2016) showed no 
significant difference in plasma glucose concentration during a 120 min OGTT 24 h after a 
passive heating bout in the water compared to a normothermic control condition. To date, the 
effects of acute passive heating in close proximity to an OGTT in individuals with T2DM has 
yet to be investigated. However, there has been promising evidence from healthy volunteers, 
showing that passive heating may be just as good or even better at reducing hyperglycaemia 
than exercise [Faulkner et al., 2017]. Faulkner et al. (2017) showed that compared to exercise, 
passive heating reduced peak plasma [glucose] significantly more and AUC plasma [glucose] 
the same amount. Additionally, there has been an abundance of animal studies showing 
beneficial effects on glycaemic control that have investigated the effect of passive heating 
[Bathaie et al., 2010; Chung et al., 2008; Gupte et al., 2009; Gupte et al., 2010; Kokura et al., 
2007; Morino et al., 2008]. Together, these results show that passive heating in close proximity 

 
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to a glucose challenge has the potential to improve glucose tolerance, insulin-stimulated 
glucose uptake and increase insulin signalling in individuals with T2DM. 
 
In contrast to warm water and its purported increased glucose disposal [Faulkner et al., 2017; 
Hooper, 1999], warm ambient conditions have previously been shown to increase plasma 
glucose concentrations. For example, plasma glucose levels increase after a 120 min OGTT 
in temperatures of 30-35℃ compared to 20-25 in healthy young individuals [Moses et al., 1997]. 
This has also been shown in individuals with diabetes (obese and non-obese) and health 
obese and non-obese [Akanji & Oputa, 1991; Akanji & Oputa, 1991]. Obesity spears to play a 
key role in the change in glucose concentration, driven by an increased heat conduction time 
[Akanji & Oputa, 1991]. 
 
Proposed mechanisms for this include, increasing cutaneous blood flow, subsequently 
reducing visceral blood flow and therefore reducing glucose disposal from the blood [Frayn et 
al., 1989; Moses et al., 1997]. However, increases in ambient air temperature are unlikely to 
increase core temperature over long periods when ambient air temperature is ≤ 28℃  
[Haldane, 1905]. Whist increases in core temperature have been strongly linked with increases 
in visceral [Rowell et al., 1968; Rowell., 1970; Rowell et al., 1971] and skeletal muscle blood 
flow [Crandall et al., 2008; Heinonen et al., 2011]. Importantly, when there is a significant 
increase in ambient air temperature muscle both muscle and liver blood flow are reduced 
[Rowell 1968]. Conversely, an increase in core temperature will perfuse visceral [Rowell et al., 
1968 Rowell., 1970; Rowell et al., 1971] and skeletal muscle tissue [Crandall et al., 2008; 
Heinonen et al., 2011], which are key sites for glucose disposal and may have a beneficial 
effect.  
 
 
Hooper (1999) postulated that an increase in skeletal muscle blood flow may be responsible 
for this increased glucose clearance, citing evidence that this can modulate insulin mediated 
glucose uptake [Baron et al., 1994]. Other mechanisms have also been suggested, but have 
yet to be confirmed,  including; increased insulin sensitivity [Hermanns et al., 2014; Raz et al., 
2009], altered inflammatory response [Hundal et al., 2002; Shoelson et al., 2003; Yuan et al., 
2001] and activation of heat shock proteins (HSP) [Rodrigues-Krause et al., 2012]. Each 
potential mechanism is discussed below: 
 
1) Increased skeletal muscle and liver blood flow:  
 
Since the hypothesis that heating may cause an elevation in blood flow and increase glucose 
clearance, several studies have looked to examine the effect of heating on blood flow. Using 
local heating, whole body heating and water perfused suites, Crandall et al., (2008) and 
Heinonen et al., (2011) showed that passive heating increases both muscle blood flow and 
cardiac output. This may be at least in part due to passive heating raising core temperature, 
therefore increasing cardiac output and also muscle blood flow in an attempt to lose heat. 
Interestingly, Baron et al. (1994) showed that an increase in plasma insulin caused a significant 
increase in leg muscle blood flow from ~0.15 L·min-1 to 0.35 L·min-1. Insulin is produced for the 

 
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vasodilatory response, which aids increased blood flow and maintains blood pressure [Baron 
et al., 1994; Heinonen et al., 2011].  
 
This increase in cardiac output and skin blood flow is not dissimilar to when individuals 
exercise in the heat [Braun et al., 1995; Brunt et al., 2016; Crandell et al., 1999; Faulkner et 
al., 2017; Heinonen et al., 2011; Rivas et al., 2016; Voulgari et al., 2013]. Therefore, passive 
heating may mimic the beneficial effects on glucose homeostasis seen following exercise. 
While this evidence supports a rise in muscle blood flow during heating, there is no evidence 
as to how long blood flow stays elevated post heating without actively cooling individuals. 
 
 
 
2) Passive heating as an exercise mimetic  
 
Multiple studies have examined the effect of exercise-induced hyperthermia and glucose 
homeostasis [Braun et al., 1995; Caldwell et al., 2011; Faulkner et al., 2017; Gibson et al., 
2014; Jorge et al., 2011; Kadoglou et al., 2007; Kang et al., 1996; O’gorman et al., 2006; 
Periard et al., 2011; Voulgari et al., 2013]. All of these studies show that exercise improves 
insulin sensitivity irrespective of the aerobic exercise programme that was used. Acute aerobic 
exercise increases muscle glucose uptake five-fold via insulin-independent mechanisms 
[Roberts et al., 2013] and even a single bout of high-intensity exercise can improve insulin 
sensitivity [Cockcroft et al., 2018]. Post-exercise, glucose uptake remains elevated via both 
insulin-independent (~120 min) and insulin-dependent (up to 48 h) mechanisms if exercise is 
prolonged [Magkos et al., 2008]. Improvements may be seen in insulin sensitivity post exercise 
after just 20 min of high-intensity exercise, lasting for ~24 h [Gillen et al., 2012; Manders et al., 
2010]. Low-intensity aerobic exercise (≥60 min) can enhance insulin sensitivity in obese, 
insulin –resistant adults for at least 24 h post exercise [Newsom et al., 2013]. This suggests 
that short, low cardiovascular strain may lead to improved insulin sensitivity.  
 
Experimental evidence of passive heat therapy is sparse, and its effects on insulin sensitivity 
has only recently been examined. However, the proposed mechanisms are similar to that 
observed in exercise trials; 1) elevated metabolic rate [Braun et al., 1995], 2) cardiac output 
[Faulkner et al., 2017; Rivas et al., 2016], 3) skeletal muscle blood flow [Heinonen et al., 2011], 
4) skin blood flow [Crandall et al., 1999] and an elevated vasodilatory response [Brunt et al., 
2016; Voulgari et al., 2013]. This suggests a potential for passive heating as an alternative to 
exercise to improve glycaemic control. Therefore, passive heating may offer an alternative 
treatment modality for individuals who cannot engage in exercise or struggle with adherence 
to exercise programmes [Ary et al., 1986; Barnard et al., 2000; Clark, 1997; Glasgow et al., 
1997; Nathan et al., 2006; Stunkard and Messick, 1985] 
 
 
3) Activation of heat shock proteins  
 

 
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Exercise-induced hyperthermia causes heat stress to cells all around the body [Periard et al., 
2011]. Heat stress triggers the production of eHSP via mRNA inside the cells in an attempt to 
protect cells from damage to extreme stress [Hayden et al., 2005]. In individuals with T2DM, 
deleterious manifestations increase in prevalence such as chronic inflammation as 
physiological stress increases [Hotamisligil, 2006; Shoelson et al., 2006]. This chronic 
inflammation (of inflammatory cytokines such as tumour necrosis factor- alpha (TNF-α)) then 
leads to impaired insulin signalling by inhibiting the IκB kinase in insulin sensitive organs 
[Hundal et al., 2002; Shoelson et al., 2003; Yuan et al., 2001]. In tissues where impaired insulin 
signalling occurs, a decrease in eHSP has been seen [Atalay et al., 2004; Bruce et al., 2003, 
Kavanagh et al., 2009] via a reduced in concentration of heat shock factor-1 [Atalay et al., 
2004; Bruce et al., 2003, Kavanagh et al., 2009]. Since eHSP is an anti-inflammatory molecule, 
when there are reduced levels of this protein, it causes increased inflammation [Hooper and 
Hooper 2009], which in turn will alter insulin function [Chung et al., 2008; Li et al., 2008]. This 
progressive cycle is illustrated in Figure 1.  
 
Acute exercise results in an upregulation of HSP mRNA (coding for eHSP), which ultimately 
leads to protection of cells by ensuring reduced accumulation of harmful protein aggregates 
(phosphorylated Tau proteins and amyloid precursors) [Hayden et al., 2005]. HSP mRNA is 
downstream of protein expression and is indicative of future protein expression [Henstridge et 
al., 2010]. The advantage of using HSP mRNA over intracellular heat shock proteins (iHSP) is 
that it can take up to 24h for the production of iHSP from stimulation [Hunter-Lavin et al., 2004]. 
However, HSP mRNA presents itself almost immediately from the initiation of stimulation 
[Henstridge et al., 2010]. Therefore, HSP mRNA can give a more valid representation of any 
acute changes that may have happened from an intervention and should also mean this 
mechanism plays a role in reducing blood plasma [glucose] after heating as well. [Bruce et al., 
2003; Chung et al., 2008; Henstridge et al., 2010 and Kurucz et al., 2002]. Surprisingly, the 
utility of measuring HSP70 mRNA is questionable given the transcription of the HSP70 gene 
(via heat shock factor) does not always result in increased protein expression [Bruce et al., 
1993; Hensold et al., 1990]. It may, therefore be prudent to measure eHSP as opposed to 
mRNA. 
 
Reduced eHSP in individuals with T2DM can be used as a predictor of the disease [Atalay et 
al., 2004, Bruce et al., 2003, Kavanagh et al., 2009]. Physiological stresses such as exercise, 
and passive exposure to heat cause an inflammatory response, which results in the production 
of eHSP [Faulkner et al., 2017].  

 
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Figure 1. Shows the interaction of changes between inflammation, impaired insulin signalling 
and heat shock proteins. Taken from Hooper, 2009. 
 
An increase in eHSP causes a reduction in inflammation by blocking the activation of 
inflammatory kinases and pro-inflammatory transcription factors through blocking the 
transcription factors targets, activation and binding [Stice and Knowlton, 2008]. A reduction in 
inflammatory status then increases insulin sensitivity by reducing the activation of serine-
threonine kinase, c-jun amino terminal kinase and inhibitor of ĸ B kinase in insulin sensitive 
organs: liver, skeletal muscle and adipose tissue [Hotamisligil, 2006; Shoelson et al., 2006]. 
C-jun amino terminal kinase and inhibitor of ĸ B kinase both impair insulin function of the insulin 
receptor and interfere with downstream insulin signalling [Hotamisligil, 2006; Shoelson et al., 
2006]. It is unknown whether this process is elicited through the mechanisms from exercise 
itself, the hyperthermia caused by exercise or a combination of both.  
 
 
 
4)   Inflammatory markers and insulin sensitivity 
 
Reducing in inflammation may provide a means to improve insulin signalling and thus improve 
in glycaemic control and insulin sensitivity [Chung et al., 2008; Li et al., 2008] in individuals 
with T2DM. Previous studies have investigated inflammatory biomarkers in individuals with 
T2DM using exercise as a treatment protocol, which show that immediately after exercise there 
is an increase in inflammatory biomarkers, which subsequently cause an increase in anti-
inflammatory biomarkers [Jorge et al., 2011; Kadoglou et al., 2007]. This data suggests that 

 
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regular exercise can result in a chronic reduction in inflammation. A potential mechanism for 
this may be that exercise increases metabolism and thus increases core and muscle 
temperature, resulting in an increased heat stress to cells in the body [Periard et al., 2011] and 
an increase in in eHSP and this then reduces inflammation [Chung et al., 2008; Li et al., 2008]. 
Faulkner et al., (2017) showed that when a healthy population underwent a passive heating 
protocol there was an immediate inflammatory response which they concluded would promote 
an anti-inflammatory response, however they also showed that 2 h post passive heating, IL-6 
inflammatory response reduced to almost baseline levels again. In contrast, any initial increase 
in inflammatory biomarkers can be used as an indicator for a longer term increase in anti-
inflammatory biomarkers [Steensberg et al., 2003]. Steenburgs et al., (2003) results showed 
that there was a delayed anti-inflammatory response ([IL-1ra] and [IL-10]) of 60 min from 
infusion of IL-6.  
 
To date, evidence of passive heating on anti-inflammatory biomarkers is scarce in individuals 
with T2DM. To date, one study has shown found that after a 6-month exercise training 
programme there was a reduction in inflammatory biomarkers (TNF-α, high sensitivity CRP, 
IL-18) and an increase in resting anti-inflammatory biomarker (IL-10) concentrations (Kadoglou 
et al., [2007]). Therefore, more data on the anti-inflammatory response to passive heating in 
individuals with T2DM is needed. 
 
5) Autonomic function 
 
Individuals with T2DM have reduced autonomic function which leads to, reduced cardiac and 
vascular function, and cardiovascular disease [Voulgari et al., 2013] which is caused at least 
in part by chronic hyperglycaemia [Kitsios et al., 2011; Papanas et al., 2011]. Chronic 
hyperglycaemia induces several metabolic pathways to undergo a repetitive vicious cycle 
which leads to micro- and macrovascular damage [Kitsios et al., 2011; Papanas et al., 2011]. 
Hot environments may, via the same physiological outcomes as exercise, cause 
cardiovascular strain and so may be a long term way of training the heart, without needing to 
exercise. This may improve the cardiovascular outcome for individuals with T2DM [Faulkner 
et al., 2017]. 
 
Simple, inexpensive interventions aimed at improving glycaemic control in individuals with 
T2DM are needed to help the growing health and economic burden. Using passive heating as 
a therapeutic strategy for maintaining glucose homeostasis, and improving metabolic and 
cardiovascular health is a novel, developing field, worthy of further investigation. Considering 
the above, there is a clear rationale to explore further whether passive heating has therapeutic 
potential in individuals with T2DM. We therefore propose to investigate the effect of passive 
heating on glucose tolerance (plasma [glucose]), plasma [insulin], insulin sensitivity, interleukin 
6 concentration [IL-6], [IL-10], [eHSP-70], HRV, muscle blood flow and thermal sensation / 
comfort. 
 

 
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5. 
PRELIMINARY STUDIES AND EXPERIENCE OF INVESTIGATORS 
Dr. Anthony Shepherd is a Senior Lecturer in Physical Activity, Exercise and Health at the 
University of Portsmouth. During his career he has worked with several clinical populations, 
including T2DM, stroke, geriatrics, Raynaud’s and scleroderma, cystic fibrosis (CF), end-
stage renal disease and chronic obstructive pulmonary disease. This work was undertaken 
at the University of Exeter Medical School, the Royal Devon and Exeter NHS Foundation 
Trust Hospital, South Devon Healthcare NHS Foundation Trust, Portsmouth Hospitals Trust 
and University Hospital Southampton where he held honorary contracts. His research has an 
overarching theme of clinical exercise physiology and therapeutic treatments. His most recent 
publications have looked at the effect of increasing the bioavailability of nitric oxide (NO) in 
clinical cohorts via nutritional supplementation. He has disseminated this work in 
internationally recognised journals and holds research grants from charities and industry 
partners.    
Mr. Thomas James completed his BSc at the University of Roehampton in Sport and Exercise 
Science. During his time there he became interested in physiology in extreme environments 
and conducted his final year project investigating the cardiovascular and thermoregulatory 
adaptations of a high-intensity interval training, short-term heat acclimation protocol in trained 
cyclists. He then attended Bangor University and completed an MRes. in Sport and Exercise 
Physiology. During his time there he focused his research more on cardiovascular physiology 
in extreme environments. His research and thesis for his MRes. investigated the autonomic 
control of cardiovascular responses to the human diving reflex. For Thomas’ PhD research, 
he will be applying this knowledge and skill base to drive research into novel and inexpensive 
T2DM treatments. More specifically, looking at the metabolic and cardiovascular health of 
individuals with T2DM while using extreme stressors (e.g. warm water) with the aim of 
developing a new, inexpensive and adherable treatment. The research has the potential to 
help reduce the economic burden on the NHS, as well as improving the well-being of 
individuals with T2DM. Mr James will be assisted by two postgraduate students, Mr Billy 
Hopkins and Mr Connor Morgan. A third year student, Miss Emily Windsor will also assist in 
data collection. All students have valid GCP certificates and have been training in all 
procedures.   
Dr. Jo Corbett has worked at the University of Portsmouth since 2005 and is currently 
Associate Head for Innovation and Impact in the Department of Sport and Exercise Science 
as well as the REF coordinator for UoA24. His PhD was awarded by the University of 
Southampton and examined pharmacological and non-pharmacological approaches to 
increasing fat metabolism. Subsequently his research has focused on environmental 
stressors. He is an established research leader in sport, exercise and occupational activities 
in hot environments with 50+ peer-reviewed publications in the field; he was lead author on 
the 2018 British Association of Sport and Exercise Sciences Expert Statement on Interventions 
for Improving Performance in the Heat. Jo is an advisor to the English Institute of Sport on 
exercise in the heat and is a British Association of Sport and Exercise Sciences accredited 
Physiologist. 

 
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Dr. Joseph Costello is a Senior Lecturer in Exercise Physiology at the University of 
Portsmouth. He completed his PhD in exercise physiology at the University of Limerick, 
Ireland. After his PhD, he moved to Queensland University of Technology (Australia) where 
he worked as a post-doctoral research fellow in the Institute of Health and Biomedical 
Innovation for three years. Joseph then undertook further post-doctoral research in the 
Extreme Environments Laboratory with the Department of Sport and Exercise Science at the 
University of Portsmouth, before being appointed as a Lecturer in 2016 and recently Senior 
Lecturer. Dr. Costello is currently on the editorial board of Experimental Physiology, the 
commissioning editor for Extreme Physiology & Medicine, and a methodological editor for the 
Cochrane Bone, Joint and Muscle Trauma Group. 
Prof. Mike Tipton joined the University of Portsmouth from University of Surrey in 1999. In 
addition to his University positions, he was based at the Institute of Naval Medicine from 1983 
to 2004 and was Consultant Head of the Environmental Medicine Division from 1996. Mike 
has spent 30 years working in the areas of thermoregulation, environmental and occupational 
physiology. Mike and is colleagues in the Extreme Environments Laboratory examine the 
physiological and psychological responses to adverse environments and the selection, 
preparation and protection of those who enter such environments. He is Director of Research 
for the Department of Sport & Exercise Science, he provides advice to a range of universities, 
government departments, industries, medical, search and rescue and media organisations. 
Prof. Janis Shute is a Professor of Pharmacology at the University of Portsmouth and is head 
of the Respiratory Immunopharmacology group, investigating mechanisms and biomarkers of 
lung inflammation and novel strategies to improve inhaled drug delivery in patients. Prof. Janis 
Shute has worked to develop novel inhibitors of neutrophil elastase, a key mediator of tissue 
damage in the lung. Other research includes the role of coagulation factors in bronchial 
epithelial damage and repair in normal and asthmatic airways; endothelial dysfunction and the 
links between inflammatory markers and oxidative stress markers. She will advise on 
biochemical analysis, processing of samples, storage of samples and aid with protocol and 
manuscript development. 
 
Prof. Michael Cummings is a Consultant in Diabetes and Endocrinology at the Portsmouth 
Hospitals Trust and is also an Honorary Professor in Diabetes and Endocrinology at the 
University of Portsmouth. His major areas of interest in diabetes are linked to cardiovascular 
disease and risks (in particular dyslipidaemia and microalbuminuria), diabetes and pregnancy 
and erectile dysfunction. His primary research interests are in endothelial dysfunction, 
oxidative stress and dysglycaemia. He secured funding and established a small laboratory to 
undertake non-invasive digital plethysmography and venous occlusion plethysmography, 
techniques used to examine endothelial function. This technique has been applied to 
examine the mechanisms of endothelial dysfunction in type 1 and T2DM. He also 
collaborated with the University of Portsmouth to develop the DOVE (Dysglycaemia, 
Oxidative stress and the Vascular Endothelium) project and laboratory, developing assay 
techniques to measure markers of oxidative stress and vascular inflammation. Additionally, 
he has collaborated with our team in the Department of Sport and Exercise Science on other 
projects. Many oral presentations/abstracts/original papers based upon this work have been 

 
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presented at national/international meetings and the findings have been accepted in peer-
reviewed journals for publication. He will bring an expert clinical opinion to help design and 
inform the protocol. 
Dr Iain Cranston is a Consultant in Diabetes and Endocrinology at the Portsmouth Hospitals 
Trust. He is also the Clinical Lead for Diabetes and Intensified Insulin Therapy, Diabetes 
inpatient care and Diabetes and Renal Disease. Iain is an expert in continuous glucose 
monitoring (CGM) and diabetes, and will help with protocol design and data analysis. He will 
bring an expert clinical opinion to help inform the protocol and will help with participant 
recruitment.  
Dr. Zoe Saynor is a Senior Lecturer in Physical Activity, Exercise and Health at the University 
of Portsmouth, whilst also holding several visiting academic positions at University College 
London and the University of Toulon. She has 10 years’ experience conducting research and 
applied work with individuals with various chronic health conditions, including CF, asthma, 
non-CF bronchiectasis, rheumatoid arthritis, renal disease, diabetes (T2DM and CF-related), 
Raynaud’s phenomenon and joint hypermobility. She has particular expertise in CF, having 
published numerous research articles and delivered several invited talks on the topic, however 
she has a varied clinical portfolio of research at present. Since her time at the University of 
Portsmouth, she has embedded clinical research within both the University Hospital 
Southampton and Queen Alexandra Hospital (Portsmouth) and has established the Clinical, 
Health and Rehabilitation research Team within the Department of Sport and Exercise Science 
at the University of Portsmouth.  
 
6. 
AIMS AND OBJECTIVES 
The global aim of this investigation is to establish if an acute bout of passive, warm water 
therapy reduce plasma [glucose] in the blood in response to an OGTT.  
6.1 
Primary Objective 
1. To determine if an acute bout of passive, warm water therapy reduces plasma 
[glucose].  
6.2 
Secondary Objectives 
To determine: 
1. whether plasma [glucose] reduces more if the passive, warm water therapy is 
conducted before or after the OGTT?  
2. whether plasma [insulin] reduces more if the passive, warm water therapy is conducted 
before or after the OGTT?  
3. whether insulin sensitivity increases following an acute bout of warm water therapy? 
4. whether fuel utilisation alters during and following an acute bout of warm water 
therapy?  
5. whether cardiovascular status is altered during or after an acute bout of warm water 
therapy?  

 
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6. whether eHSP increases during an acute bout of warm water therapy? 
7. whether inflammatory status reduces during or after an acute bout of warm water 
therapy? 
6.3 
Summary of Study Design 
Recruitment 
Participants will be recruited from adult diabetes outpatient clinics at the Queen Alexandra 
Hospital (Portsmouth), via a database of known individuals with T2DM from the Wessex, 
Diabetes, National Institute for Health Research (NIHR) Clinical Research Network and via 
posters and word of mouth at the University of Portsmouth. Only males and post-menopausal 
women will qualify for the study. Finally, the local Desmond diabetes awareness group 
meetings will be used to identify potential volunteers. Potential participants will either be 
approached by their T2DM consultant, the principal investigator, member of the research team 
or a research nurse. Following the participants declaration of interest, via telephone, mail, 
email or in clinic, they will be given a participant information sheet. A member of the research 
team will subsequently approach the participant ≥ 48 h later, to answer any questions and 
organise consent, screening and familiarisation (visit 1).  
The design will be a balanced, randomised, within-subject comparison of three conditions 
(condition 1: OGTT in 23 oC air, condition 2: OGTT 30 min before 60 min in ~39oC water (~ 
90% RH), condition 3: OGTT 30 min after 60 min in ~39°C water (~ 90% RH)). There will be 4 
visits in total, visit 1 is for consent, screening and familiarisation and visits 2, 3 and 4 are 
experimental conditions.  
 
Visit 1 (consent, screening and familiarisation)  
During visit 1, participants will give their informed consent, followed by a health screening 
questionnaire. In addition to the health screening questionnaire, medical history and a blood 
sample will be collected and analysed for a full blood count, glycated haemoglobin (HbA1c), 
liver and kidney function. Finally, a resting electrocardiogram (ECG) will also be recorded and 
then examined for irregularities, where a clinical decision will be made on further participation 
to the study by consultants at QA hospital. Participants will then be shown the rest of the 
equipment and taken through the procedure for the next 3 visits and, if the participant is happy 
to continue the study, the next visit will be organised.  
 
Pre experimental instructions  
Before all experimental visits the participants will be instructed to keep their normal diet up to 
48 h before coming in for their visit and also replicate it before each visit (they will be given a 
food diary). They will be asked to avoid exercise and hot baths / saunas 48 h before each visit 
(can have brief (< 5 minutes) luke-warm showers) and consume 17 mL.kg-1 water (for 
examples an 80 kg male would need to drink at least 1.36L of water) and other fluids ad libitum 
throughout the active day (~ 16 h) [Benelam and Wyness, 2010] and 4 mL.kg-1 upon waking 
the morning of the visit. All conditions will require the participant to fast for 12 h before coming 

 
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to the start of the visit. For the pre-test diet, participant will be asked to keep a normal balanced 
diet (they will record their intake in a diary). Participants will also be instructed on suitable 
clothing to wear for each visit (explained again in the PIS). In line with OGTT guidelines, 
participants will be asked to not take their diabetes drugs on the morning of the test 
(hypoglycaemic agents before the fasting period are to be taken as normal). Prior to leaving 
the laboratory, participants will be provided with lunch, then asked to take their tablets and 
blood sugars checked prior to leaving.  
 
Visit 2, 3 and 4 
Participants will arrive at the laboratory at ~9 am for conditions 1 and 2 and 8 am for condition 
3. Prior to a 15 min resting period (supine) before any measures are taken participants will be 
asked to insert a rectal thermistor (participants will be given clear instructions using our SoPs). 
Condition 1, 2 and 3 will be balanced and participants randomly allocated to begin the study 
in either visit 2, 3 or 4 using a blinded member of the team.  
For all visits (see figure 2), participants will lie in a semi recumbent position in minimal clothing 
(bathing shorts and a t-shirt) for the entirety of the visit. Initially, participants will be cannulated 
(Versatus winged and ported IV cannula, Terumo, Japan) and blood samples drawn for 
analysis of osmolality (Lithium Heparin (LH) tubes BD, USA) plasma [glucose] 
(Fluoride/Oxalate tubes, BD, USA), [insulin] (Ethylenediaminetetraacetic acid (EDTA) K2, BD, 
USA), and [eHSP70] (EDTA K2, BD, USA) at baseline and every 30 min of each experimental 
visit. Following cannulation an 180 min OGTT (75g) (Rapilose OGTT solution, Penlan 
healthcare, Japan) will commence in a thermoneutral room (~ 23oC). A maximum of 18 mL of 
blood being drawn at each time point (max 126 mL per visit). To maintain the patency of the 
cannula and to reduce the risk of infection, after every sample is taken, 5 mL of saline will be 
flushed through the cannula. Then before every sample is taken, 2.5 mL of blood will be drawn 
out of the cannula to ensure any remaining saline will not interfere with the samples and data 
interpretation (additional 17.5mL per visit). During the OGTT, HR (via electrocardiogram) will 
be measured continuously, whilst blood pressure (M5-1, Omron, Japan), deep body 
temperature (rectal probe) and resting metabolic rate (indirect calorimetry) (Quark CPET, 
Cosmed, Italy) will be assessed every 30 min.  
Condition 2 will employ identical procedures to condition 1, except thirty minutes into the 
OGTT, the participant will be immersed into an immersion tank (~39oC) for 60 min. Water 
temperature will be manipulated as required to achieve and maintain a target Trec at 38.5 oC 
using water between 37.5 and 39oC, and then participants will be removed horizontally back 
into the thermoneutral room for the reminder of the OGTT. Participants will be towel dried and 
given a towelled robe to wear. Condition 3 will employ identical procedures to condition 2, with 
the exception that the heating via immersion will start as soon as the participant is 
instrumented (and following a 15 min rest period) and the OGTT will commence 30 min after 
the 60 min immersion time for a further 180 min (see figure 2 for a schematic). Finally, if willing 
participants will be asked to take part in a brief exit interview about their experience.  
 

 
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Figure 2. Schematic of the 3 experimental conditions. BP, thermal sensation, HRV, osmolality, insulin sensitivity, 
RMR, eHSP70, IL-6, IL-10, plasma glucose and insulin measured at 30 min intervals from 0930-1230 for visit 1 and 
2, and from 1000-1300 for condition 3 (with the addition of a baseline measure at 0830 for visit 3). Muscle 
oxygenation at 0930, 1000, 1130, 1200 and 1230 for condition 1 and 2. Muscle oxygenation at 0830 and from 1000-
1300 at 30 min intervals for condition 3. HR continuous for entirety of visit. Solid line indicates immersion period 
and dashed line indicates OGTT period. 
 
6.4 
Primary and Secondary Endpoints/Outcome Measures 
1. To determine if an acute bout of passive, warm water therapy reduces plasma 
[glucose].  
 
Secondary outcomes are to determine: 
1. whether plasma [glucose] reduces more if the passive, warm water therapy is 
conducted before or after the OGTT?  
2. whether plasma [insulin] reduces more if the passive, warm water therapy is conducted 
before or after the OGTT?  
3. whether insulin sensitivity increases following an acute bout of warm water therapy? 
4. whether fuel utilisation alters during and following an acute bout of warm water 
therapy?  
5. whether HRV increases during or after an acute bout of warm water therapy?  
6. whether eHSP increases during an acute bout of warm water therapy? 
7. whether inflammatory status reduces during or after an acute bout of warm water 
therapy? 
 
 
 

 
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7. 
STUDY PARTICIPANTS 
7.1 
Study Setting 
Consent, screening and familiarisation will commence at the diabetes centre and Queen 
Alexandra hospital. All experimental visits will take place in the Extreme Environments 
Laboratory at the University of Portsmouth has a 30-year history of conducting innovative 
human research trials. The laboratory contains world leading facilities for conducting 
environmental research including three bespoke environmental chambers, with temperature, 
humidity, simulated solar, wind and hypoxia control, and a research group involving over 20 
academics and researchers.  
 
7.2 
Overall Description of Study Participants 
Adults with a clinical diagnosis of T2DM, as defined by the WHO. A standard medical history 
and clinical examination will be undertaken by a research nurse or a member of the research 
team. Information taken during the examination will be height, weight, full blood count 
(analysed from a blood sample that will be collected), HbA1c, neuropathy severity and a resting 
ECG. ECG will be examined for irregularities and a clinical decision will be made on further 
participation to the study by a consultant at QA hospital. 
7.3 
Eligibility Criteria 
Inclusion Criteria 
The participants must meet ALL of the following criteria to be considered eligible for the study: 
● 
Male or female (either post-menopausal or in the early-follicular phase (3-5 days after the 
onset of menstruation) of the menstrual cycle), aged 35 years or above. 
● 
Diagnosed with T2DM as defined by the WHO.   
● 
Participant is willing and able to give informed consent for participation in the study. 
● 
Participant is able to understand and fully cooperate with the study protocol. 
 
Exclusion Criteria 
The participant may not enter / be withdrawn from the study if ANY of the following apply: 
● Severe peripheral neuropathy (to the point to which they cannot sense temperature) 
● Uncontrolled hypertension (≥180 systolic / 100 diastolic mmHg) 
● Taking any medication which may interfere with data interpretation or safety  
● Who have had a myocardial infarction or cerebro−vascular event 
● Any cardiac abnormalities which restrict hard exercise  
● Current smokers or who have stopped within 3 months 
● Participant is unable to understand and/or fully cooperate with the study protocol 

 
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● Any other serious medical condition which would interfere with data interpretation or safety 
will be excluded from participation. 
● Any skin conditions including ulcerations  
8. 
SAMPLING 
Plasma glucose AUC is our primary outcome. No study to date has assessed the effect of hot 
water immersion on glucose tolerance in individuals with T2DM. Therefore, no data were 
available to power our primary outcome. For 90% power with an α-level set at P < 0.05 (two 
tailed), to detect a 1 SD difference, 13 volunteers are required to compare within group for 
placebo and active conditions. In order to counterbalance our design, 18 participants will be 
required to complete the testing. Therefore, we aimed to recruit 20 individuals with T2DM to 
account for a 10% drop-out rate typically seen in our clinical trials. Resultant effect sizes will 
be used to power future trials.  
9. 
STUDY PROCEDURES 
9.1    Recruitment 
Participants will be recruited from adult diabetes outpatient clinics at the Queen Alexandra 
Hospital (Portsmouth), via a database of known individuals with T2DM from the Wessex, 
Diabetes, National Institute for Health Research (NIHR) Clinical Research Network and via 
posters and word of mouth at the University of Portsmouth. Additionally, participants will be 
recruited through primary care and via posters. Finally, the local Desmond diabetes awareness 
group meetings will be used to identify potential volunteers. Potential participants will either be 
approached by their T2DM consultant, the principal investigator, member of the research team 
or a research nurse. Following the participants declaration of interest, via telephone, mail, 
email or in clinic, they will be given a participant information sheet. A member of the research 
team will subsequently approach the participant ≥ 48 h later, to answer any questions and 
organise consent, screening and familiarisation (visit 1). A maximum payment of £10 will be 
available per visit upon request. 
9.2   Screening and Enrolment 
Consent and screening will occur at the diabetes centre at Queen Alexandra hospital. A 
standard medical history and clinical examination will be undertaken by a research nurse or a 
member of the research team at the screening visit (visit 1) after consent has been taken, 
including height (stadiometer, Seca, Germany), weight (Ohaus, USA), ECG and blood sample 
collection for lipids and biochemical markers, urea and electrolytes, HbA1c and liver function 
tests.  
Blood pressure: Semi recumbent resting blood pressure: measurements will be performed on 
the morning of the screening visit; serial measurements on each participant will be made at 
the same time of day and same arm for each visit. The recordings will be made in quiet, 
comfortable, ambient laboratory conditions. Measurements will be made with a 
semi−automated device conforming strictly to European Hypertension Society (as adopted by 

 
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the British Hypertension Society) and American Heart Association guidelines. Five 
measurements will be made and the final three will used to determine an average blood 
pressure. 
9.3 Randomisation 
An online computer programme will be used to randomly allocate and counterbalance 
participants to being the trial in one of three conditions. This will be performed by a blinded 
member of the research team.  
 
9.4 Study Assessments 
Pre-trial instructions 
Participants will be instructed to keep their normal diet and to keep it the same 48 h before 
each trial (they will record their intake in a diary). Additionally, they will have to fast for 12 h 
before the start of each visit. Participants will be instructed to consume 17 mL.kg-1 of water 
and other fluids ad libitum throughout the active day (~16 h) the day before each visit [Benelam 
and Wyness, 2010] and 300 mL upon waking the morning of the visit. Participants will also be 
told to bring a bathing suit and a t-shirt to wear for the experimental visits. Additionally, 
participants will also be told that they cannot perform exercise or have a hot bath / sauna 48 h 
before each visit (can have brief (< 5 min) luke-warm showers). Visits to the laboratory should 
be made either via car or public transport and should not be made via physical activity e.g. 
cycling. Parking will be provided. At the start of each visit, plasma osmolality will be assessed 
via freezing point depression (model 3320 osmometer, advance instruments, USA). In line with 
OGTT guidelines, participants will be asked to not take their diabetes drugs on the morning of 
the test (hypoglycaemic agents before the fasting period are to be taken as normal). Prior to 
leaving the laboratory, participants will be provided with lunch, then asked to take their tablets 
and blood sugars checked prior to leaving.  
 
Passive heating and Core Temp 
The passive heating conditions (conditions 2 and 3) will be conducted in a thermoneutral room 
(~ 23°C air, ~ 90% RH), at the same time of day, with the heating taking place for 60 min in a 
heated pool (starting at 39°C water). Pre and post body mass will also be recorded, along with 
any fluid or solids going into or out of the body. Participants will firstly be cannulated lying 
horizontal, then will lie semi-recumbent and baseline measures taken. Depending on which 
condition (either 2 or 3) the OGTT or the passive heating will start first. Passive heating will 
start with the participant entering the pool and lying semi-recumbent on a submerged bed with 
their head, neck and cannulated arm above the water. Participants’ rectal temperature (Trec) 
will be monitored so that once it reaches ~38°C the water will be cooled to ~ 38 - 38.5°C so 
that it will maintain Trec at ~ 38.5°C (Trec is still expected to rise slightly after reduction in water 
temperature, see below for more details). If Trec reaches 39°C or above at any point, the 
participant will immediately be withdrawn from the study and a cooling protocol administered 
(see SOP attached). Additionally, if systolic BP raises above 180 mmHg or HR above 140 

 
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bpm, participants will be immediately withdrawn from the study. Passive heating will cease by 
winching the participant, still lying semi-recumbent, out of the immersion tank and onto a semi-
recumbent bed. The participant will then be towel dried and given a blanket (to ensure 
maximum heat retention) and remain semi-recumbent until completion of the visit. The 
reminder of the visit will be conducted in the thermoneutral room.   
Trec will be measured continuously throughout each visit using disposable rectal thermistors 
(Rectal temperature probe, Philips, Netherlands). Participants will self-insert the thermistor to 
15 cm (this will be marked with sterilised tape) into the rectum using standard operating 
procedures. Trec will be closely monitored throughout the passive heating conditions to ensure 
that it does not exceed 39°C.  
 
OGTT and Insulin Sensitivity  
Insulin sensitivity will be calculated using values from both plasma glucose and plasma insulin. 
Multiple calculations for insulin sensitivity exist, each with their own strengths and weaknesses. 
Calculations used can be split into two categories: fasting indices and OGTT indices. A meta-
analysis [Otten et al., 2014] of all the current surrogate measures of insulin sensitivity showed 
that the most valid fasting calculation to use compared with the gold standard HEC method, 
was the quantitative insulin sensitivity check index (QUICKI). Out of the OGTT surrogate 
insulin sensitivity measures Otten et al. (2014) highlighted 4 indices that were the most suitable 
surrogate for HEC: Stumvoll, OGIS, Matsuda and Gutt. 
Following an overnight fast, blood samples will be taken for the measurement of fasting 
glucose and insulin levels, and this data will be used to calculate β-cell function via the QUICKI 
model [Katz et al., 2000], 1/[Log(I0)+Log(G0)] (I=insulin, G=glucose, 0=baseline) which has 
been shown to have a strong positive correlation with the HEC [Otten et al.,2014]. Post-
prandial glycaemic control will be measured by a 180 min OGTT (maximum of 75 g anhydrous 
glucose), with blood samples taken from a cannula at 0, 30, 60, 90, 120, 150 and 180 min of 
the OGTT, with β-cell function being calculated using the Stumvoll method (Equation 1).  
 
0.226-0.0032xBMI-0.0000645xI120-0.00375xG90  
Equation 1. Stumvoll’s insulin sensitivity index equation [Stumvoll et al., 2000] 
 
Thermal sensation / comfort  
Both thermal comfort and sensation will be measured using a 20 cm scale (0 = very 
cold/uncomfortable; 10 = neutral; 20 = very hot/comfortable; modified from Zhang et al., (2004) 
and recorded prior to immersion, during immersion and following the 60 min immersion, at 15 
mins intervals.  
Resting Metabolic Rate (metabolite utilization)  

 
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Breath-by-breath changes in pulmonary gas exchange and ventilation will be non-invasively 
measured at rest to enable the estimation of resting metabolic rate (RMR). The assessment 
of pulmonary gas exchange (e.g. Vo2 and Vco2) will give us the respiratory exchange ratio 
(RER). RER is a reliable and valid measure of fuel utilisation. To assess RMR, participants will 
be asked to lie down and rest for 15 mins, following this, pulmonary gas exchange will be 
measured via a gas analysis system for 5 min, this will be repeated every 30 min until the end 
of the visit. Participants will be required to use a mouth piece with a nose clip. In order to 
assess inspired and expired gas (volume and concentration), gases will be sampled through 
a capillary line attached to the face mask. A sub analysis of this breath by breath data will give 
us the respiratory exchange ratio (a value of 0.7 indicates only fatty acid metabolism and 
values of > 1 indicates only glucose metabolism, any value in the middle equates to a 
combination of both). This is an accurate assessment of rate of energy expenditure and fuel 
utilisation, and will be indicative of what proportion of carbohydrate and fat is being 
metabolised [Blond et al., 2011]. In order to calculate the rate of carbohydrate and fat 
metabolism the Frayn (1983) equations will be used. Pulmonary gas analysis will be performed 
on all testing visits. Calibration will follow the guidelines given in the user manual produced by 
COSMED Omnia. Briefly, this involves calibrating the gas analyser by passing through a 
sample of gas with a known concentration (Oxygen: 16%, Carbon dioxide: 5%, Nitrogen: Bal) 
and calibrating the volume on the gas flowmeter by using a 3 L syringe to pass a known volume 
of air through the flowmeter. 
 
Near- infrared spectroscopy  (NIRS) 
Muscle oxygenation in the microcirculation of the m. vastus lateralis will be measured using a 
commercially available NIRS system (Portamon, Artinis, Netherlands) during the pre- and 
post-immersion periods. This system is non-invasive and will be placed on m. vastus lateralis 
of the right leg. NIRS is used to estimate changes, relative to baseline, in oxygenated and 
deoxygenated haemoglobin concentrations, myoglobin concentrations, total haemoglobin and 
tissue saturation index to give an insight into the dynamic balance between muscle O₂ delivery 
and utilisation. Furthermore, a skin-fold measurement at the site in which the NIRS device is 
fixed will be recorded, in line with the manufacturer’s recommendations. 
 
HR and BP 
HR will be automatically calculated from the ECG, real-time. Blood pressure will be measured 
every 30 min via a patient monitor brachial BP cuff (Dynascope, Fukuda Denshi, Japan). 
 
HRV 
HRV will be calculated as a low frequency (LF) variable and root mean square of successive 
differences (RMSSD). LF has been chosen as it indicates a physiological origin of both 
sympathetic and vagal activity. RMSSD is then being calculated additionally as it gives a 
stronger representation of vagal tone (due to it being affected much less by respiratory sinus 

 
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arrhythmia (RSA)) than LF thus improving validity of the vagal tone measure [Laborde et al., 
2017]. Both measures will be calculated from an ECG trace. 
 
Biochemistry  
Venous blood samples will be collected via cannulas (ideally located in the Cephalic vein on 
the forearm, alternatively ether in hand or antecubital fossa. All locations will out of the water 
at all times) into fluoride, LH and EDTA vacutainers during all visits. All whole blood will be 
centrifuged at 4500 x g for 10 mins, at 4 °C and aliquoted plasma will be frozen at -80 °C for 
subsequent analysis. 
Plasma [glucose] will be determined by the Department of Biochemistry at the Queens 
Alexandra Hospital, Portsmouth. Commercially available enzyme-linked immunosorbent 
assays will be used to determine [insulin], [IL-6], [IL-10] and [eHSP70]. Haemogloin and 
haematocrit will be measured via Haemacue (Hb 201, Sweden) and capillary tubes 
(Microhaematocrit capillary tubes, Hawksley, UK) respectively at all data collection time points 
in triplicate. 
 
Follow-up questions 
A few semi-structure follow-up questions will be given to the participants to get feedback on 
how they felt in the hot water in both conditions. The questions will be related to how they felt 
in terms of heat tolerance, lethargy, well-being and also how they felt in the hours and days 
following each visit.  
9.5 
Discontinuation/Withdrawal of Participants from Study Treatment 
Participants may be withdrawn from the study if they ask to withdraw, if participant safety is in 
question or if the study is stopped early.   
9.6 
Definition of End of Study  
The end of study will be following the final dissemination of results, which is expected to be 
during September 2019.  
 
10. 
INTERVENTIONS 
10.1 
Description of Study Intervention / Treatment 
The passive heating condition will be conducted in a thermoneutral room (~ 23°C air, ~ 90% 
RH) with the heating taking place for 60 min in a heated pool (starting at 39°C water (~ 90% 
RH) and then cooling to clamp Trec at 38.5°C). The participant will firstly be cannulated, lie 
horizontal and baseline measures taken after 15 min rest period. Following baseline measures 
an OGTT will start. Thirty min into the OGTT, the passive heating will start with the participant 
entering the pool and lying horizontally on a submerged bed with their head and neck elevated 

 
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above the water. Participants’ rectal temperature (Trec) will be monitored so that once it reaches 
38°C the water will be cooled slightly to arrest the rise in Trec and maintain Trec at 38.5°C (Trec 
is still expected to rise slightly after reduction in water temperature, see below for more details). 
If Trec at any point reaches 39°C or above, the participant will immediately be withdrawn from 
the study and a cooling protocol administered (see SOP attached). Passive heating will cease 
by winching the participant out of the pool and onto the dry bed with the participant still lying in 
a semi-recumbent position. The participant will then be dried and given a blanket (to ensure 
heat retention as would be the case with putting on clothing after bathing) and remain semi-
recumbent until completion of the visit. The reminder of the OGTT will be conducted in the 
thermoneutral room (~23°C room temperature).  
Core temperature (Trec) will be measured continuously throughout each visit using disposable 
rectal thermistors (which will be checked for accuracy in a water bath prior to each trial ± 
0.2°C). Participants will insert the thermistor 15 cm (this will be marked with sterilised tape) 
into the rectum using standard operating procedures. Trec will be closely monitored throughout 
the passive heating condition to ensure that it does not exceed 39°C. For analysis, data will 
be grouped into 1 min average intervals (and will be collected at a 1 Hz sampling frequency).  
10.2     Adherence to Study Treatment 
Participants will be immersed into the pool and core temperature measured to ensure safety. 
Participants can, at any time, ask to be safely removed from the pool. Additionally, participant 
will be removed from the pool if Trec reaches >39°C. Total time in the pool will be calculated 
for each participant on each visit.  
10.3     Accountability of the Study Treatment 
Any of the study investigators are able to be accountable for maintaining the 
appropriate water temperature.  
10.4     Concomitant Medication / Therapies 
Participant involvement is anticipated to be 3 morning sessions within a period of ~3 weeks. 
Medication will be recorded on enrolment and updated throughout their time on the trial. For 
each experimental visit, hypoglycaemic aids will be stopped for the morning (see pre 
experimental instructions for details).   
 
11. 
ASSESSMENT OF SAFETY 
11.1 
Definitions  
A serious adverse event (SAE) is any untoward medical occurrence that: 
● Results in death 
● Is life-threatening* 

 
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*The term "life-threatening" refers to an event in which the participant was at risk of death; 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 
● 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 SAE when, based upon appropriate medical judgement, 
the event may jeopardise the participant and may require medical or surgical intervention to 
prevent one of the outcomes listed above. 
11.2 
Reporting Procedures for Serious Adverse Events 
SAE occurring to participant should be reported to the research ethics committee (REC) that 
gave a favourable opinion of the study where in the opinion of the Chief Investigator the event 
was: ‘related’ – that is, it resulted from administration of any of the research procedures; and 
‘unexpected’ – that is, the type of event is not listed in the protocol as an expected occurrence. 
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the national research ethics service (NRES) 
report of serious adverse event form (see NRES website).  
11.3 
Recording and Reporting Procedures for All Adverse Events 
All adverse events will be recorded in participant file notes. Distinction between SAE and 
adverse event (AE) will be made by an independent medical officer. All SAE will be reported 
to the sponsor and the REC regardless of if it is related or not.   
   
12. 
DATA HANDLING AND RECORD KEEPING 
12.1 
Data Collection Forms 
Visit 1 will act as a screening and consent (ECG checked by clinical physician and blood to be 
analysed for a full blood count, HbA1c, estimated glomerular filtration rate (eGFR) and liver 
function). All measures for this visit will be stored in the clinical records folder (CRF). Separate 
forms will be prepared for; anthropometrics, medical history, screening (i.e. blood pressures 
etc) and blood markers. On subsequent visits data forms will be used to collect temperatures 
of the labs and immersion pool. All paper copies will be stored in the CRF’s.   
12.2     Data Management 
All data will be double data entered into excel. Macro’s will be used to check for anomalies and 
corrected. The participants will be identified by a study specific participants number and/or 
code in any database.  The name and any other identifying detail will not be included in any 

 
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study data electronic file. The Chief and Principal Investigators are responsible for database 
maintenance and management.  
13. 
DATA ANALYSIS 
13.1      Description of Analysis Populations  
Participants who completed all 4 visits will be entered into the analysis. 
13.2      Analysis of Endpoints  
All data will be tested for normality. Normally distributed data will be presented as means ± 
SD. Statistical analyses will be performed on SPSS software version 24.0 (Chicago, IL).For 
normally distributed data a one-way analysis of variance (ANOVA) will be used to identify 
differences between conditions for plasma [glucose] (AUC and peak), plasma [insulin], insulin 
sensitivity, [eHSP70], [IL-6], [IL-10], HRV, NIRS and RMR. Where significance differences are 
observed, post hoc test will then be performed. Where data is not normally distributed, 
appropriate non parametric tests will be utilised.  
13.3     Procedure for Dealing with Missing, Unused and Spurious Data  
Outliers will remain within the data. Within the data analysis software missing data will be 
coded 9999 and the data point will be missed during analysis. 
13.4     Procedures for Reporting any Deviation(s) from the Original Statistical Analysis 
Plan 
For this study a statistical analysis plan is not required (excluding the above). 
13.5      Interim analysis and criteria for early study termination  
Due to the small samples size and time frame, no interim analysis will be performed. Early 
termination will only occur if the safety of participants is in question.  
 
14. 
ETHICS 
NHS Local Research Ethics Committee (REC) approval will be obtained prior to 
commencement of the study. The REC, local NHS, research and development department 
and all site specific forms and patient identification centre forms will be forwarded to the R&D 
department at Queen Alexandra Hospital Portsmouth, prior to recruitment of participants. 
Written informed consent will be obtained from all participants. Insurance indemnity will be 
provided by the University of Portsmouth for this study.  
Every effort has been made to keep the risks and discomforts to a minimum, with first aid cover 
and extensive screening procedures in place but there are some risks associated to taking 
part. Not all volunteers will experience any or all of the risks stated below, but participants will 
be made aware of them. 

 
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The main burden to participants is a 12 h fasting period before their visit. Participants will be 
asked to cease all hypoglycaemic agents on the morning of testing and will be instructed to 
bring them with them on the day. This is in line with a normal fasted OGTT typically used in 
clinics. Prior to leaving the laboratory, participants will be provided with lunch, they can take 
their tablets and blood sugars checked prior to leaving.  
We will also be asking people with T2DM to be submerged in hot water for condition 2 and 3. 
This may cause some discomfort which will be assessed with a thermal sensation and comfort 
scale. We will be measuring the core temperature via rectal thermistor and monitoring it closely 
at all times. Participants will be reminded that they are free to withdraw at any time point and 
will also be withdrawn and cooled if Trec raises above 39°C (as stated in the departments 
SAP’s). 
Participants will be asked to insert a rectal thermistor on two out of the four visits (water 
immersions). This can be uncomfortable upon insertion; however, this is necessary for safety 
purposes.  Participants will be provided with lubrication and instructions.  
Other less burdensome tests will be performed such as:  
Blood pressure; participants will feel a cuff squeeze their arm and this can be uncomfortable 
for a few moments. Blood samples taken every 30min via cannulation (using the appropriate 
hygienic technique and by a trained individual): Intravenous access can cause discomfort 
associated with insertion of the needle. Infusion of saline (5 mL.30min-1) will be applied to 
minimise infection risk (flushing and locking eliminates potential nesting sites for 
microorganisms) and clotting. To avoid the saline affecting subsequent samples, 2.5 mL of 
blood will be drawn from the cannula before they are collected. 
Other risks that have been accounted for are hypotension and loss of consciousness in the 
water. If at any point the participants BP is measured at ≥ 180/100 the trial will be terminated 
and the participant withdrawn from the study. If there is loss of consciousness in the water the 
participant will immediately be harnessed securely out. This can be done quickly as they will 
already be lying on the sling. Once they are out of the water and placed on a bed, their 
breathing will be checked and first aid administered. They will also be cooled via cold water 
spray and a fan. Additionally, their head will never go underwater as their head will be elevated 
above waterline throughout all submersions.   
14.1 
Participant Confidentiality 
The study staff will ensure that the participants’ anonymity is maintained. The participants will 
be identified only by initials and a participants ID number on the CRF and any electronic 
database.  All documents will be stored securely and only accessible by study staff and 
authorised personnel. The study will comply with the Data Protection Act which requires data 
to be anonymised as soon as it is practical to do so.   
 
14.2     Other Ethical Considerations 
N/A  
 

 
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14.3     Declaration of Helsinki 
The study protocol will be carried out in accordance with the Declaration of Helsinki. 
14.4     ICH Guidelines for Good Clinical Practice 
All research staff exposed to participants will be good clinical practice (GCP) trained and will be 
monitored by the sponsor to ensure adherence to GCP. 
 
15. 
PATIENT PUBLIC INVOLVEMENT (PPI) 
15.1    Study design 
Evidence of patient, carer and public involvement (PCPI):  
The chief investigator has a strong track record of PCPI work having been involved in PCPI 
award winning projects recently from the UK Stroke Forum (October 2016) for another 
acceptability and feasibility trial. The lay summary and patient facing documentation has been 
reviewed by our PCPI co-investigator.      
15.2    Study implementation 
Issues will be managed by the senior members of the research team.  
15.3    Dissemination 
We endeavour to disseminate findings through local groups, local/national media, research 
papers and international conferences.  
 
16. 
FINANCING AND INSURANCE 
16.1    Research Costs 
The majority of consumables and personnel required for testing have been funded by the 
University of Portsmouth. All hardware and software is already available. 
16.2    Service Support Costs 
No service support costs are being sought. 
16.3    Excess Treatment Costs 
No excess treatment cost are being sought. 
16.4    Study Sponsorship  
This study is being sponsored by the University of Portsmouth. 
 

 
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17. 
TIMETABLE AND ORGANISATIONAL CHART 
February 2018: Funding for consumables has been secured 
July 2018: Application for ethical approval 
October 2018 - June 2019: Participant recruitment 
October 2018 - April 2020: Data collection 
April 2020 – June 2020: Data analysis 
June 2020: Study completion date 
Post-June 2020: Dissemination of results through conference proceedings, journal 
submissions and practitioner outlets.   
18. 
RESOURCES, EQUIPMENT AND PHYSICAL FACILITIES 
In addition to the departmental support, and in order to facilitate the completion of the work, 
the research team will have full access to all of the necessary world-leading extreme 
environment facilities that are housed within the department estate given that they are pivotal 
to the success of the project. Specifically, this will enable temperature control within the 
facilities to ensure reproducibility. Finally, the department will ensure that full technical 
assistance is provided to enable the set-up of the laboratories and testing rooms.  
19. 
DISSEMINATION AND OUTCOME 
Results (estimates of effect sizes and confidence intervals) from this study will be utilised to 
define our primary outcome and to power a larger more complex study. The research is to be 
disseminated in internationally recognised, peer reviewed journals and at relevant scientific 
conferences, as well as contributing to Mr. Thomas James’ PhD thesis. Furthermore, all 
screening data will be utilised as part of the patient’s annual report, and distributed to clinicians 
and participants upon request.  
20. 
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21. 
APPENDIX 1 
    SCHEDULE OF PROCEDURES 
22. 
APPENDIX 2    STUDY FLOW CHART 
23. 
APPENDIX 3    PARTICIPANT INFORMATION SHEET 
PUT PHT TEMPLATE HERE 
24. 
APPENDIX 4    INFORMED CONSENT FORM 
PUT PHT TEMPLATE HERE 

 
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25. 
APPENDIX 5    SAMPLE QUESTIONNAIRES 
26. 
APPENDIX 6    SAMPLE DATA COLLECTION FORMS 
27. 
APPENDIX 7    DRUG INFORMATION (SUMMARY OF PRODUCT 
CHARACTERISTICS)  
28. 
APPENDIX 8 MANUFACTURERS BROCHURE FOR NOVEL 
EQUIPMENT  
29. 
APPENDIX 9 CONTRACTUAL AGREEMENTS WITH OUTSIDE 
CONSULTANTS / COLLABORATORS / INSTITUTIONS (E.G. 
INDUSTRY, CONTRACT RESEARCH ORGANISATIONS)