Effect of Short-term Negative Energy Balance With or Without Exercise on Systemic Biomarkers in Patients With Knee Osteoarthritis
Phase: NA
Conditions: Knee Osteoarthritis
Sponsor: University of Bath

REC reference: 20/SW/0063                     IRAS ID: 271897                               Version 2.0, 13/10/2020  
 
 
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Protocol: Effect of short-term negative energy balance with 
or without exercise on systemic biomarkers in patients with 
knee osteoarthritis  
REC reference: 20/SW/0063 
 
INVESTIGATORS  
Professor James Bilzon 
Dr Enhad Chowdhury  
Ms Rachel Deere 
Mr Matthew Farrow 
Professor Dylan Thompson  
Dr Fiona Watt 
Dr Ben Davies 
 
 
ADDRESS 
Department for health 
University of Bath 
Bath 
BA27AY 
 
Correspondence: J.Bilzon@bath.ac.uk  
 
 
 
 

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Contents 
 
SUMMARY .................................................................................................................................................. - 3 - 
SYSTEMIC BIOMARKERS IN OSTEOARTHRITIS ............................................................................................................. - 4 - 
IMPACT OF DIET AND EXERCISE ON SYSTEMIC BIOMARKERS ASSOCIATED WITH OSTEOARTHRITIS .......................................... - 6 - 
PURPOSE OF STUDY ............................................................................................................................................. - 6 - 
PARTICIPANTS ............................................................................................................................................ - 7 - 
INCLUSION AND EXCLUSION CRITERIA ...................................................................................................................... - 7 - 
RECRUITMENT .................................................................................................................................................... - 8 - 
SAMPLE SIZE ...................................................................................................................................................... - 8 - 
GROUP ALLOCATION/RANDOMISATION ................................................................................................................... - 9 - 
OUTCOME MEASURES ................................................................................................................................ - 9 - 
PRIMARY OUTCOME VARIABLE ............................................................................................................................... - 9 - 
SECONDARY OUTCOME VARIABLES .......................................................................................................................... - 9 - 
STUDY OVERVIEW ...................................................................................................................................... - 10 - 
STUDY DESIGN ........................................................................................................................................... - 10 - 
PRE-SCREEN ..................................................................................................................................................... - 10 - 
PRELIMINARY TESTING ....................................................................................................................................... - 10 - 
PRE-TRIAL VISITS ............................................................................................................................................... - 11 - 
TRIAL VISITS ..................................................................................................................................................... - 11 - 
WASH-IN PERIOD .............................................................................................................................................. - 11 - 
INTERVENTION GROUP 1 .................................................................................................................................... - 11 - 
INTERVENTION GROUP 2 .................................................................................................................................... - 12 - 
PROCEDURES ................................................................................................................................................... - 14 - 
Blood sampling ........................................................................................................................................ - 14 - 
LIFESTYLE INTERVENTION .................................................................................................................................... - 14 - 
Diet .......................................................................................................................................................... - 14 - 
Exercise.................................................................................................................................................... - 14 - 
DISADVANTAGES/RISK AND DISCOMFORT ................................................................................................ - 14 - 
METHODS .................................................................................................................................................. - 16 - 
BLOOD STORAGE ............................................................................................................................................... - 16 - 
FUNCTIONAL TESTS ............................................................................................................................................ - 16 - 
SELF-REPORT OUTCOMES .................................................................................................................................... - 16 - 
INDIRECT CALORIMETRY ..................................................................................................................................... - 16 - 
EXPERIMENTAL PAIN MEASURES ........................................................................................................................... - 17 - 
STORAGE AND MANAGEMENT OF DATA AND CONFIDENTIAL INFORMATION ........................................... - 17 - 
REWARDS AND FEEDBACK ......................................................................................................................... - 17 - 
REFERENCES ............................................................................................................................................... - 18 - 
 
 
 
 
 
 
 

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Summary 
Osteoarthritis (OA) is the most prevalent form of arthritis and one of the leading causes of 
chronic disability worldwide [1]. OA is now considered to be a low-grade inflammatory disease 
[2] characterised by the progressive degradation and loss of articular cartilage. Diagnosis and 
progression of OA has traditionally been the product of radiography, however more recently, 
biological markers (biomarkers) are being increasingly explored. Biomarkers are direct 
indicators of biological, pathogenic and pharmacological responses to interventions and 
represent acute changes in tissue adaptations long before such changes are evident in 
radiographic images of biological structures, particularly the cartilage [3]. Early identification 
of disease therefore offers a window of opportunity for therapeutic intervention. 
 
Long-term weight loss studies consisting of diet and exercise manipulation have highlighted 
beneficial effects on inflammation and clinical outcomes in overweight persons both with [4] 
and without OA [5], and 3-4 weeks of exercise exposure has been shown to positively influence 
pain and function in those with symptomatic knee OA[6]. We hypothesise that by creating a 
short-term negative energy balance through a combination of caloric restriction and exercise 
[7] that we will be able to reduce inflammation in individuals with knee OA. We also aim to 
determine if a dietary energy restriction condition can also reduce inflammation. We will 
examine changes in systemic biomarkers associated with inflammation in those with early-
stage knee OA in response to the short-term interventions. We will also assess the impact of 
the intervention upon self-reported measures of pain/function and performance in functional 
tests. Importantly, we will be able to determine any of these effects without the confounding 
factor of substantial weight loss present in longer-term interventions.  
 
As well as tracking symptomatic pain, it is also relevant that we incorporate a range of 
experiential pain measures. Such measures are used to assess pain sensitisation which is a 
phenomenon reported in individuals who suffer with knee OA [8]. Pain sensitisation in the 
knee OA population is suggested to be caused by peripheral inflammation at the joint and 
systemic level altering the way that the central nervous system responds to tactile and 
noxious stimuli [9]. 

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The study will include both male and female participants that fit the eligibility criteria. 
Participants’ activity levels and dietary intake will be monitored for 7 days and each will then 
be randomised into 1 of 2 intervention groups. Both groups will complete a 4-week 
observational wash-in period for us to determine the stability of the biomarkers, with 
participants acting as their own controls. Both intervention groups will then reduce their 
caloric intake by 5000kcal/week over 4 weeks, and the second intervention group will have 
the addition of 5 moderate-vigorous intensity exercise sessions (based upon target ratings of 
perceived exertion) per week for 30 minutes using a cycle ergometer to determine the added 
effects of exercise.  
 
Systemic biomarkers in Osteoarthritis 
Despite evidence of improved patient related/clinical outcomes (i.e. pain and function) in 
knee OA research, changes in radiographic images fail to show in a large percentage of 
patients that take part in clinical trials, even those lasting up to 3 years [10]. This emphasises 
the need to develop biomarkers that are both sensitive to change and predictive of disease 
activity to enhance such trials, improve the area of OA phenotyping and to allow for an overall 
improvement in patient care with regard to disease management. Additionally, systemic 
rather than local (within the joint i.e., articular cartilage, synovial fluid) biomarkers are less 
invasive and easier to acquire. 
 
Cartilage degeneration is a hallmark of OA and is attributed to several catabolic mediators 
including ADAMTS5, MMP-13, hypoxia-inducible factor 2 and complement component 5+. 
Of these, MMP-13 (Matrix Metallopeptidase-13) has attracted the most interest and focus 
due to its significant over-expression in OA joints, with almost no detection in normal joint 
tissues [11]. MMP-13 acts primarily on type II collagen [12] but also on aggrecan [13]. 
 
The most influential group of biomarkers associated with inflammation in knee OA are well 
known but not limited to; IL-1β, TNF-α, IL-6, IL-17, and IL-18. These biomarkers act 
catabolically on the homeostasis of tissues forming the joints and are associated with the 
expression of matrix metalloproteinase (MMPs) (primarily MMP-1; 3 and 13)[14]. As previously 
detailed, MMP-13 has been the main enzyme from the MMPs family to be attributed to 
cartilage degeneration by acting on both type II collagen and aggrecan. MMP-1 and MMP-3 

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are also potentially relevant with MMP-1 suggested to participate in osteochondral changes 
seen in OA as determined by the expression of MMP-1 from subchondral bone cyst cells [15]. 
MMP-3 has been positively related to the severity of knee OA, with its expression being 
greater with increased severity [16]  but its specific role in knee OA progression is unclear.   
 
Biomarkers that are associated with MMP-13 expression, act either directly, or indirectly and 
either by inhibiting or increasing its expression. Two of the most influential cytokines 
associated with the increased expression of MMP-13 in in-vitro OA cartilage are IL-1 and 
TNF-   . On the other hand, IL-10, IL-4 and IL-13 are associated with cartilage repair and 
suppress/inhibit the expression of MMP-13 through IL-1 and TNF- [19, 20] during in-vitro 
studies. However, there lacks evidence to support these findings in-vivo. Many of those 
biomarkers associated with MMP-13 expression have also been associated with knee pain 
and function.  IL-1β [21]  and TNF-α [22] have been positively associated with high WOMAC 
scores for each sub scale of pain, stiffness and physical function. Conversely, an infusion of IL-
4-10 protein improves deficient joint loading in animals[23], used as a surrogate marker of pain. 
Although there is no direct evidence highlighting the relationship between IL-13 and clinical 
outcomes, it is associated with supressing IL-1β and TNF-α which as stated above, are both 
positively associated with increased knee pain and reduced function.  
 
As well as those inflammatory biomarkers associated with MMP-13 expression, it is also 
essential to highlight and focus attention on the influence of two key biomarkers associated 
with the pathogenesis of knee OA: CRP and IL-6. CRP is a marker of low grade systemic 
inflammation and is well known to be positively associated with cartilage volume, disease 
progression [24] and local joint inflammation[25]. IL-6 is a primary regulator of CRP but is also 
directly associated with knee cartilage loss [26] and is a significant predictor of knee OA [27], but 
it is unclear of the role of either in the pathogenesis of knee OA. 
 
Focusing on IL-6, CRP, IL-1β, and TNF-α, IL-4, IL-10 and IL-13 allows us to target biomarkers 
linked with both clinical outcome measures (i.e., pain and function) and early-stage disease 
progression (i.e. inflammation and cartilage degeneration).  
 

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Impact of diet and exercise on systemic biomarkers associated with Osteoarthritis.  
Diet and exercise treatments are widely recommended through clinical guidelines with the 
aim of improving pain and functional ability [28], and it is well documented that the 
manipulation of diet and/or exercise, have beneficial effects on systemic inflammation [29, 30], 
especially in those who are overweight/obese.  Despite this, the current scope of research 
around the effects of diet only and combined diet and exercise treatments on OA biomarkers 
lacks in both the number of available studies, and the biomarkers assessed in those studies 
[31]. Additionally, those studies that have induced a negative energy balance through diet and 
exercise manipulation have been long-term investigations whereas our study is focused on 
relatively short-term changes. This has the potential benefit of improving systemic 
inflammation while being relatively independent of the effect of substantial weight change 
(i.e. loss of >5kg often seen in longer term studies), which is known to be a critical factor that 
reduces systemic inflammation[32] and can also independently improve pain and functional 
outcomes. 
 
Purpose of study 
The primary aim of the study is to assess changes in the primary clinical biomarker of systemic 
inflammation (CRP) in individuals with knee osteoarthritis. 
 
We hypothesise that a 4-week lifestyle intervention employing a negative energy balance will 
decrease systemic CRP concentrations.  
 
Secondary outcome variables – 
- 
Changes in systemic pro- and anti-inflammatory cytokines and chemokines associated 
with knee OA.  
- 
Changes in fasted markers of metabolic function (glucose, NEFA, triglycerides, 
total/HDL cholesterol)  
- 
Changes in self-reported functional and pain outcomes 
- 
Changes in functional tests 
- 
Changes in experimental pain measurements  

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Participants  
Eligibility for participation in this research trial will be based on information and 
measurements gathered at a preliminary screening visit.  
 
We will recruit participants based on the following eligibility criteria: 
• Diagnosed with uni -or bi-lateral knee osteoarthritis. Clinically deemed to be 
appropriate for conservative treatment and not surgical intervention through Virgin 
Care.   
• Male or females with clinically diagnosed and radiographically confirmed knee 
osteoarthritis. Exclusion of other possible causes of knee pain/arthritis determined 
based upon clinical reasoning. 
• Aged between 45 to 69 years. 
• Body mass index of ≥27.5 kg/m2 (to ensure we are working with chronic, unresolved 
low grade inflammation not just the result of injury – those with a BMI of >25kg/m2 
have  clinically raised levels of CRP compared with those with a BMI of <25kg/m2 [33] ). 
• Average daily physical activity level (PAL - total energy expenditure/resting energy 
expenditure) of <2.00, to ensure the individual is not highly active.  
• Have no physical impairment unrelated to the knee that would prevent safe 
participation and their ability to comfortably complete cycling exercise under sub-
maximal conditions. 
 
Inclusion and exclusion criteria  
Inclusion 
Exclusion  
Male or post-menopausal females  
Inability to undertake cycling exercise safely** 
Clinically 
diagnosed 
with 
knee 
Osteoarthritis 
including radiographic confirmation 
Use of prescribed anti-inflammatory medication*** 
Oxford Knee Score ≥20 to ≤35 for index knee  
Current smoker [34] (or having quit <6months ago)  
Aged 45-69 years 
Has a diagnosis of diabetes or other metabolic 
disorder 
BMI ≥27.5kg/m2 to ≤40.0 kg/m2 
Use of other medications that might interfere with 
study outcomes 
Daily physical activity level <2.00* 
Recent (within last 3 months) change in body mass 
of >5% 

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Currently engaged in an ongoing programme of 
physiotherapy treatment 
 
Currently on waiting list or having had surgery for 
Osteoarthritis on the index knee (prior OA related 
surgery permitted on other knee if this is greater 
than 12 months prior to enrolment) 
 
Having had major hip/knee surgery within preceding 
12 months 
 
Individuals with a blood pressure of greater than 
180/110 
 
Currently pregnant or intending to become pregnant 
during the study 
*Assessed during a preliminary period of ≥7 days via accelerometer. 
**Assessed during pre-screening via physical activity readiness questionnaire (PAR-Q+). 
***Patients that occasionally use NSAIDS will be asked to use paracetamol as a ‘rescue medication’ if they 
experience pain during the trial, with a minimum of a 3-day washout period prior to the trial. 
 
Recruitment  
 
Patients will be recruited through Virgin Care acting as a patient identification centre: 
1) Potentially eligible patients attending a Health Optimisation clinic for Osteoarthritis 
(for individuals with a BMI >30 kg/m2  or current smokers) as part of services provided 
by Virgin Care will be provided with study information. These individuals have been 
previously clinically diagnosed with knee osteoarthritis by a physiotherapist or general 
practitioner before being referred to the Health Optimisation Clinic. Physiotherapists 
or exercise instructors delivering these classes will provide study information 
(Participant Information Sheet) to potentially relevant individuals based upon age, 
body mass and Oxford Knee Score as described in inclusion criteria above.  
 
As part of this identification process, only the direct care team will interact with the patients 
to the point at which individuals are provided with study information. 
 
Sample size  
A previous intervention conducted in sedentary overweight and obese women using the same 
energy restriction model for 3 weeks[7], established an effect size of 1.22 for the change in 
CRP with dietary energy restriction and vigorous intensity exercise 5 x per week. Using this 
effect size, an α of 0.05, and a power of 80%, it is estimated that a sample size of 8 would be 

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required for a two-tailed paired comparison. However, as we would hypothesise that the 
effect of the diet only intervention would be less, due to the lesser energy restriction and 
independent impact of exercise training upon CRP, an estimated effect size of 0.85 is 
proposed. Using an α of 0.05, and a power of 80%, it is estimated that a sample size of 13 
would be required for a two-tailed paired t-test. Assuming a dropout rate of ~25%, we would 
aim to recruit ~16 individuals in each intervention arm. Between-group comparisons will be 
exploratory in nature, to determine sample sizes of future studies comparing the efficacy of 
the conditions.         
 
Group allocation/Randomisation 
Following screening/baseline measures of those included, participants will be allocated to 
either intervention group 1 (Diet restriction) or intervention group 2 (Diet restriction + 
Exercise) by an individual outside of the research team who will have no interaction with 
participants. To protect against chance imbalance between the two groups, this will be 
conducted via minimisation accounting for participant sex, age, BMI, and Oxford Knee Score.  
Outcome measures 
Primary outcome variable 
- 
Change in the primary clinical biomarker of systemic inflammation (CRP). 
 
Secondary outcome variables  
- 
Changes in in systemic pro- and anti-inflammatory cytokines and chemokines 
associated with knee OA. 
- 
Markers of metabolic function (glucose, NEFA, triglycerides, total/HDL cholesterol)  
- 
Self-report measures of physical function and knee related symptoms 
- 
Physical Activity Level 
- 
Functional tests 
- 
Pain measurements  

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Study overview 
 
 
Figure 1. Study Schematic - General overview (detailed information on page 13) 
 
Study design 
Pre-screen 
After potential participants have been recruited, they will be invited to the University of Bath 
for eligibility assessment and pre-screening following the provision of written informed 
consent. We will determine if individuals meet all inclusion criteria or any of the exclusion 
criteria through a screening questionnaire and measurement of BMI and resting blood 
pressure. We will also ask individuals to undertake the Oxford Knee Score questionnaire to 
allow us to use this data as part of our participant allocation. Participants will each be fitted 
with a physical activity monitor (ActiheartTM) attached to the chest with a strap or 2 adhesive 
ECG pads.  Participants will wear this physical activity monitor for 7 days. This data will allow 
us to calculate participant physical activity level (PAL) to ensure they meet the inclusion 
criteria for this measurement (i.e., <2.00).  
 
Preliminary testing 
The purpose of preliminary testing is to allow us to characterise the sample population based 
on anthropometric and fitness characteristics. We will note participant’s body mass (kg), 
height (cm) and BMI (kg/m2) and ask them to complete a submaximal exercise test on a cycle 
ergometer with expired gas analysis to predict maximal aerobic capacity. This will allow us to 
predict HRmax and VO2max so that we can estimate the intensity at which individuals have been 
working during their training periods.  
 

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Pre-trial visits 
In the 24 hours before trial days, participants will be required to avoid exercise and alcohol 
consumption. They will also be asked to record their food and drink intake in the 24 hours 
before the first trial and repeat this prior to subsequent visits. Participants will be asked to 
arrive at the laboratory fasted for ≥10hrs in the morning. Along with university policy, we will 
also call participants 24hrs before their visit to complete the COVID-19 Research Participant 
Screening Form. 
 
Trial visits 
All participants (regardless of intervention group) will attend 3 separate trial visits in which 
the following measurements/tests will be performed.  
 
- 
DEXA scan + anthropometrics 
- 
Resting metabolic rate (RMR) 
- 
Venous blood sample (for collection of plasma and serum) 
- 
Functional tests 
- 
Self-report measures 
- 
Experimental pain measurements 
 
Wash-in period  
Following trial visit 1, participants’ will complete a four-week wash-in period (allowing 
participants to act as their own control). Throughout the wash in period, participants will be 
asked not to make any substantial changes to their normal lifestyle. Food diaries and activity 
monitoring will also be completed for 1-week of the wash-in period to enable prescription of 
the energy restriction diet. At the end of this 4-week period, participants will return for 
another trial visit to determine the stability of biomarkers and other outcomes under stable 
lifestyle conditions. 
 
Intervention group 1  
For the 4-week intervention period, participants in intervention group 1, will reduce their 
habitual diet by 5000 kcal per week, prescribed individually based on their 7-day food diary 

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and physical activity monitoring (i.e., energy intake and energy expenditure). The diet 
restriction will be based on an absolute energy deficit achieved by the intake of smaller food 
portions. This will be subtracted from their energy expenditure (ActiheartTM) rather than 
energy intake to avoid issues with participants’ under-reporting food intake. We will induce 
this energy deficit by multiplying the weight of all individual foods from the 7-day diary by a 
factor gathered from the energy expenditure data, allowing us to determine the desired 
energy intake for the intervention period. 
 
Intervention group 2 
For the 4-week intervention period, as per the diet alone group, participants in intervention 
group 2 will reduce their habitual diet by 5000 kcal per week. However, there will be the 
addition of five, 30-minute aerobic exercise sessions per week. Each participant will be 
provided with a cycle ergometer which will be set up in the participants home, where all 
exercise training sessions will take place. Due to the progressive nature of the exercise 
increasing weekly, researchers will check-in with participants weekly via video/telephone call 
to ensure there are no issues. Participants will also be required to wear a HR monitor for at 
least 1 exercise session a week for us to determine that the intensity is increasing weekly.  
 
Exercise sessions will be based upon individuals’ perception of intensity using the 6-20 rating 
of perceived exertion (RPE) scale (where 6 = no exertion and 20 = maximal exertion). The 
target intensity based upon RPE will progressively increase throughout the duration of the 
training. During week 1 of intervention, the target intensity will be an RPE of 12 for 30 
minutes, RPE of 13 for week 2, RPE of 14 for week 3 and RPE of 15 during week 4. These target 
intensities span the range from moderate-intensity exercise to the lower boundary of 
vigorous-intensity exercise.  

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Figure 2. Detailed schematic of the two trial periods. 

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Procedures  
Blood sampling 
Blood samples of 20ml will be collected via venepuncture using local procedures, processed 
by centrifugation immediately (plasma samples) or left to clot for 30 minutes then processed 
(serum samples). In both cases, samples will be aliquoted into 500 microlitre cryovials and 
stored in ultralow temperature freezers at -80°C until use. Trial specific Instructions will cover 
laboratory processing of samples. Measurements of biomarkers will be by established 
laboratory assays, either immunoassay or electrochemiluminescence (Mesoscale Discovery), 
previously validated for use with plasma or serum, Watt Laboratory, Oxford; with CRP, 
glucose, NEFA and total/HDL cholesterol analysed using a clinical chemistry analyser at the 
Human Physiology laboratories at the University of Bath.  
 
Lifestyle intervention  
Diet  
Based on their habitual dietary choices, participants’ calorie intake will be reduced by 
5000kcal.week-1 relative to measured energy expenditure during the week. The dietary 
restriction will be based on an absolute energy deficit achieved by the intake of smaller food 
portions. 
 
Exercise 
Participants will take part in 5 moderate-vigorous intensity (based upon RPE) stationary 
cycling exercise sessions per week for 30 minutes. This is in line with weekly 
recommendations of exercise for adults.  
 
Disadvantages/risk and discomfort  
Participants will be asked to give up their time, for pre-screening preliminary testing and 3 
laboratory visits at the University of Bath. This might pose an inconvenience for some people.  
 
Venous blood samples will be collected by venepuncture which can cause minor discomfort 
during the procedure and may result in minor bruising. In rare cases, there is the chance of 

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infection, though the occurrence of such events are further minimised by our strict 
institutional adherence to best practice and standard operating procedures.   
 
Assessment and clearance for submaximal exercise will be determined by Physiotherapists 
employed by Virgin Care as part of the participant identification process.  This will reduce the 
risk of adverse events during exercise testing. Based on participants’ choice, exercise sessions 
will be subsequently conducted at the laboratories at the University of Bath or at home with 
equipment provided by the researchers – this option will be subject to the participants 
confirming that an additional individual will be available to be present during home-based 
training. 
 
Symptoms in response to the prescribed exercise will be monitored throughout the duration 
of the intervention. Patients will be asked to rate pain in response to exercise using a 0-10 
numerical rating scale. Scores greater than 6 during or after exercise will result in some 
reduction in intensity of exercise [35]. Additionally, should elevated joint pain persist for 
greater than 24 hours than the pre-exercise level, then the intensity of the subsequent session 
will be reduced. This will be continued until symptoms subside.  
 
We will measure body composition and visceral adipose tissue in participants on the three 
trial days using a DEXA scanner. A DEXA scanner is a non-invasive radiographic technique that 
uses very low radiation (total protocol dose of 0.03 mSv) which is equivalent to experiencing 
a few days of average natural background radiation in the UK. 
 
In line with current government guidelines and university policy surrounding COVID-19, strict 
social distancing measures will be in place when possible, and where not possible, suitable 
PPE will be provided for researchers and participants. If the participant or researcher present 
with any COVID-19 symptoms during the period of the research project, they will be required 
to quarantine in line with Government advice. Further, for the collection of expired gas during 
exercise, Douglas bags will be fitted with filters to prevent the potential spread of COVID-19. 
All lab spaces/research areas will be thoroughly cleaned and sanitized between each 
participant visit. 

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The above will be explained to the potential participant verbally and in the participant information sheet to 
ensure that they are fully informed before giving consent. 
Methods 
Blood storage 
Serum blood samples will sit upright at room temperature for a minimum of 30 minutes and 
a maximum of 60 minutes before being centrifuged to allow a clot to form. Centrifugation will 
take place in a horizontal rotor for 20 minutes at 1800 g at room temperature. After 
centrifugation, we will pipette 500µl of the serum into cryovials which will then be stored at 
least -80°C in alarmed freezers. If samples are required to be moved after freezing, they will 
be shipped on dry ice to prevent thawing and re-freezing. 
 
Functional tests 
Participants will undertake the core set of functional tests recommended by OARSI: 
• A 30-s chair stand test. 
• A 40m fast paced walk test. 
• A stair climb test (consisting of 11 stairs of 16cm height). 
• Timed up and go test. 
 
Self-report outcomes 
As per recommendations[36], self-report measures will be completed after conducting of 
functional testing. The following measures will be obtained: 
• The 42-item Knee Injury and Osteoarthritis Outcome Score (KOOS). 
• The EQ-5D. 
• Short Form Health Survey (SF-36). 
 
Indirect Calorimetry 
For the calculation of resting metabolic rate, indirect calorimetry will be undertaken. 
Participants will have expired air collected using Douglas bag system and analysed for 
fractions of expired oxygen and carbon dioxide, accounting for the inspired air composition[37] 
and utilising the equations of Frayn (1983)[38] and Jeukendrup and Wallis (2005)[39] for energy 
expenditure estimation.  
 

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Experimental pain measures 
We will determine the mechanical detection threshold (MDT), pressure pain threshold (PPT) 
and dynamic mechanical allodynia (ALL) via the following methods: 
- 
The mechanical detection threshold (MDT) will be measured with von Frey filaments 
that exert forces between 0.25 and 512mN onto the surface of the skin upon bending. 
The stimulus is applied using the method of limits. That is, the stimuli are presented 
from the lowest to the highest intensity. We will determine 5 thresholds, each with a 
series of ascending and descending stimulus intensities.  
 
- 
Pressure pain threshold (PPT) will be measured using a handheld algometer. The 
stimuli will be applied to the skin at a rate of approximately 0.5 N per second. 
Participants will be instructed to verbally signal when the pressure turns to pain.  
 
- 
Dynamic mechanical allodynia (ALL) will be measured using a brush as a light tactile 
stimulator acting as moving innocuous stimuli: exerting ~200-400nM of force. This will 
be applied with a single length stroke of ~2cm over the skin. 
 
Storage and management of data and confidential information  
Blood samples will be partially processed/analysed on trial days under an anonymised 
participant identification number and then kept locked away in the laboratory, at -80°C, which 
will only be accessible to the researchers involved. After analysis, samples will be stored until 
the work has been published. Any published work from the study will include no identifiable 
participant information. Participant information and results from the study will be stored in 
an Excel data sheet, located, and maintained on a university computer, locked with a 
password shared only between the research team.  
Rewards and feedback 
As part of the intervention period, if allocated to the exercise group, participants will be given 
a cycle ergometer for use in their own home for the duration of the study period. Individuals 
in the diet only group will be offered a cycle ergometer for one month and an initial 
supervised exercise session after the completion of the study.  

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The two intervention arms are designed to produce health benefits to the participants, 
including improved aerobic capacity and potentially reduced knee pain and increased knee 
function as a result of the exercise intervention. Individuals in the diet only group would be 
expected to lose a small amount of body mass. Individualised feedback documents will be 
given to all participants, which will highlight improvements that could made to their daily 
routines i.e., diet and exercise. This document will also include information gathered from all 
tests i.e., body composition, fitness level and blood tests.  
 
References  
1. 
Cross, M., et al., The global burden of hip and knee osteoarthritis: estimates from the 
global burden of disease 2010 study. Annals of the rheumatic diseases, 2014. 73(7): p. 
1323-1330. 
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