The Effects of Types of Fruits and Vegetables on Vascular Function in Prehypertensive Participants: a Pilot Study
Phase: NA
Conditions: Blood Pressure, Vascular Stiffness, Cardiovascular Risk Factor
Sponsor: University of Cambridge

 
 
 
 
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PROTOCOL 
 
 
 
 
 
 
 
 
 
 
 
 
The effects of types of fruits and vegetables on 
vascular function in prehypertensive participants 
(CIRCUS): a pilot study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Main Sponsor 
Funders 
IRAS ID:                                                         
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
University of Cambridge  
 
Academy of Medical Sciences 
195365 
 

 
 
 
 
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Protocol authorised by: 
 
 
Dr. Linda M. Oude Griep 
 
University of Cambridge Senior Research Associate 
University of Cambridge 
MRC Epidemiology Unit 
University of Cambridge School of Clinical Medicine 
Box 285 Institute of Metabolic Science 
Cambridge 
CB2 0QQ 
  
Professor Paul Elliott 
 
Chair in Epidemiology and Public Health Medicine 
Head of Department of Epidemiology and Biostatistics 
School of Public Health 
Department of Epidemiology and Biostatistics 
St Mary’s Campus, Imperial College London 
1st Floor, room 151, Faculty of Medicine Building 
Norfolk Place, London 
W2 1PG 
 
 
Professor Gary Frost 
 
Chair in Nutrition and Dietetics 
Department of Investigative Medicine 
Hammersmith Hospital Campus, Imperial College London 
6th Floor, Commonwealth Building 
Du Cane Road, London 
W12 0NN 
 
 
Professor Elaine Holmes 
 
Chair in Chemical Biology 
Head of Division of Computational and Systems Medicine 
Department of Surgery & Cancer 
Faculty of Medicine 
South Kensington Campus, Imperial College London 
6th Floor, room 661, Sir Alexander Fleming Building 
Prince Consort Road, London 
SW7 2BB                                         

 
 
 
 
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Short title: Effects of types of fruits and vegetables on vascular function (CIRCUS) 
 
 
Long title: The effects of types of fruits and vegetables on vascular function in prehypertensive 
participants: a pilot study 
 
Study Management Group 
Chief Investigator 
Dr Linda Oude Griep 
Co-investigators 
Professor Gary Frost 
Professor Paul Elliott 
Professor Elaine Holmes 
Study Management 
Dr Linda Oude Griep 
Professor Gary Frost 
 
 
 
Sponsor 
 
The University of Cambridge is the main research sponsor for this study. For further information regarding 
the sponsorship conditions, please contact the Research Governance Manager at: 
 
Research Governance Office 
University of Cambridge, 
School of Clinical Medicine 
Box 111, Cambridge Biomedical Campus 
Cambridge, CB2 0SP 
 
 
 
 
 
 
 
 
 
PROBLEMS RELATED TO THIS TRIAL SHOULD BE REFERRED TO DR. LINDA OUDE GRIEP 
 
l.oude-griep@mrc-epid.cam.ac.uk 
 
 
 
 
 
 
 
 
 

 
 
 
 
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TABLE OF CONTENTS 
 
1. STUDY SUMMARY 
 
2. INTRODUCTION 
 
3. STUDY DESIGN 
 
4. PARTICIPANT ENTRY 
PRE-RANDOMISATION EVALUATIONS 
INCLUSION CRITERIA 
 
EXCLUSION CRITERIA 
WITHDRAWAL CRITERIA 
 
5. PHARMACOVIGILANCE 
DEFINITIONS REPORTING 
PROCEDURES 
 
6. STATISTICS AND DATA ANALYSIS 
 
7. REGULATORY ISSUES 
 
8. STUDY MANAGEMENT 
 
9. PUBLICATION POLICY 
 
10. REFERENCES 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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1. STUDY SUMMARY 
 
 
AIM 
 
 
To evaluate the effects of increased intakes of citrus fruits and cruciferous vegetables 
on vascular function in untreated, prehypertensive participants 
 
DESIGN 
 
Randomised controlled, cross-over trial with three 14-day dietary intervention periods, 
 
preceded by one week run-in period and separated by one week wash-out periods, 9 
 
weeks in total. The diet will be fully controlled and provided by the researchers. 
 
Participants will receive in random order three interventions: 
 
1: 1 portion of fruits plus 1 portion of vegetables, of commonly consumed types per 
 
day. Intakes are at 25th percentile of UK consumption and will exclude citrus fruits, 
 
cruciferous and allium vegetables. 
 
2: 4 portions of fruits plus 4 portions of vegetables, of commonly consumed types 
 
per day excluding citrus fruits, cruciferous and allium vegetables. 
 
3: 4 portions of citrus fruits plus 4 portions of cruciferous vegetables per day. 
 
Numbers of fruit and vegetable portions for the treatment groups are according to 
 
recommendations of the Dietary Approach to Stop Hypertension (DASH) diet.1 Diet and 
 
macronutrient balance will be based on UK average habitual diet (NDNS). Body weight will 
 
be maintained by tailoring the diet to individual energy requirements. 
 
POPULATION 
 
36 apparently healthy men and women, aged 40 to 65 years, with upper levels of 
 
prehypertension (systolic BP 125-160 mmHg), not using antihypertensive, other 
 
cardiovascular, or diabetic medication, with a body mass index between 20 and 35 kg/m2, 
 
with a habitual dietary pattern low in plant foods (≤4.2 portions per day) and with sufficient 
 
available food storage at home will be recruited. 
 
Exclusion criteria include smoking, pre-existing morbidity including diabetes 
 
mellitus, hypertension, cardiovascular or other metabolic diseases, ≥10 hours per week of 
 
moderate to vigorous physical activity, excessive habitual alcohol intake of >21 units per 
 
week (females) or >28 units per week, adherence to a prescribed diet, pregnancy or 
 
lactation, use of dietary supplements, food sensitivities, or vegetarian/vegan diet by choice. 
 
TREATMENT 
 
7-day run-in/washout periods: Participants will be asked to consume the run-in or 
 
washout (control) diet for 7 days. A run-in period is to minimize the effect of habitual diet 
 
intake before start of intervention, a washout period is to ensure that potential 
 
cardiometabolic effect of the previous intervention diet is eliminated. 
 
14-day dietary intervention periods: Participants will consume the control or intervention 
 
diet, weekly delivered at their home. Participants will be asked to visit the Cambridge 
Epidemiology & Clinical Trials Unit testing sites 7 times in 
total (every 1st and 15th day of each intervention sequence) in fasted condition. 
 
Day 1 and day 15 of 14-day dietary intervention periods: Participants will be asked to 
 
arrive in fasted condition and stay at the testing site until the last measurement has been 
 
completed. Participants will bring 24-hr urine and stool collections. The following 
 
measurements will be done: anthropometrics, bioelectrical impedance, blood pressure, 

 
 
 
 
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pulse wave velocity, and cognitive function. Samples of fasting blood will be taken. 
 
Participants will complete questionnaires to assess maintenance of lifestyle factors e.g. 
 
physical activity and appetite profiles throughout the study. Breakfast will be provided. 
 
DURATION 
Participants will be involved for 9 weeks in total: 
 
Week 1: Run-in period 
 
Week 2-3: 14-day dietary intervention 
 
Week 4: Wash-out period 
 
Week 5-6: 14-day dietary intervention 
 
Week 7: Wash-out period 
 
Week 8-9: 14-day dietary intervention 
 
OUTCOME MEASURES 
 
Primary outcome: Changes in systolic and diastolic blood pressure (mmHg) 
 
Secondary outcomes: Changes in: 
 
 Arterial stiffness assessed by pulse wave velocity and pulse wave analysis

 Cardiovascular risk factors measured in fasted blood samples

 Markers of endothelial function measured in fasted blood samples

 Markers of low-grade inflammation measured in fasted blood samples

 Urinary and circulatory metabolite profiles and individual metabolite levels

 Established objective urinary and circulatory markers of intakes of 
fruits, vegetables, and other foods

 Composition of gut microbiota

 Cognitive function

 Average total, daytime, night time 24-hr ambulatory blood pressure (in subsample)

 Average objective physical activity intensity and sleep time (in subsample)
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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2. INTRODUCTION 
 
2.1 Background 
 
Elevated blood pressure (BP) is a leading population-wide modifiable risk factor of cardiovascular diseases 
(CVD), which contributes yearly to ~31% of all deaths globally.2 Hypertension (≥140/90 mmHg3,4) is 
prevalent in 40% of adults, while a billion of individuals have prehypertension (120-139/80-89 mmHg3) and 
are at higher risk to develop hypertension and CVD. The strong, well-established evidence on the adverse 
relationship between higher BP and risk of CVD events emphasizes the need for targeted, practical lifestyle 
modifications to maintain healthy BP levels to prevent future CVD events. 
 
2.2 Rationale of current study 
 
Low fruit and vegetable consumption have been identified as major risk factors contributing to 
hypertension and CVD. Meta-analyses of prospective cohort studies showed strong evidence that eating 
higher amounts of fruit and vegetables lowers the risk of CVD by ~20% in a dose-response manner.5-8 Two 
landmark large intervention studies demonstrated that untreated mildly hypertensive participants who 
increased their daily fruit and vegetable intakes significantly reduced their average systolic BP by 2.8 mmHg 
systolic and diastolic by 1.1 mmHg compared to the control group with average US diet, with stronger BP-
lowering effects in hypertensive participants. 1,9 These findings underlie the evidence of the current dietary 
recommendations to eat at least 5 (80 gram) portions of a variety of fruits and vegetables per day with a 
limit of 150 ml 100% pure, unsweetened juice counting as one portion. 
 
Fruits and vegetables are however biologically and chemically complex and vary largely in amounts and 
types of nutrients and bioactive compounds. Prospective cohort studies have only since 2010 started to 
investigate the influence of eating higher amount of specific types of fruits and vegetables on the risk of 
CVD. Meta-analyses of prospective cohort studies indicate that higher intakes of apples and pears, citrus 
fruits, carrots, cruciferous vegetables (e.g. broccoli and cabbage), grapes, green leafy vegetables, raw 
vegetables, and tomatoes have been suggested to lower the risk of CVD by 14-22%.8 Few cross-sectional10 
and prospective cohort studies11,12 have been published on associations of types of fruits and vegetables 
with BP or risk of hypertension, with inconsistent findings. 
 
Subsequent dietary intervention studies attempted to demonstrate BP-lowering effects of specific types of 
fruit and vegetables, but with inconsistent findings. So far, interventions have been mainly focused on fruits 
and vegetables abundant in a single nutrient e.g., flavonoids13, lycopene14, potassium15, or nitrate16. 
Acknowledging the complexity of foods, we have only recently understood that we need to look at whole 
foods as the constituents act in combination and in synergy on health.17 Only by investigating the whole 
food will improve our understanding of potential synergistic and underlying mechanisms of BP-lowering 
effects of specific types of fruits and vegetables, which can be used to develop understandable, targeted 
dietary strategies to lower BP at a population-wide level. 
 
The main limitation of nutritional research is the use of traditional, self-reported dietary assessment 
methods with high prevalence of misreport leading to over- or underestimation of dietary intakes and 
associations and thus misinterpretation of findings. Established biomarkers to assess fruit and vegetable 
intakes include urinary potassium and plasma carotenoids, flavonoids, and vitamin C. These biomarkers 
measure intakes of nutrients from various dietary sources and are nowadays often supplemented to foods, 
which may overestimate calculation of fruit and vegetable consumption. A new approach is metabolomic 
profiling of biofluids using Nuclear Magnetic Resonance (NMR) spectroscopy and/or mass spectrometry 
(MS) to measure many small compounds in biological fluids ( urine and blood) simultaneously. Recent 
advances demonstrated that metabolomic strategies can be used to develop objective biomarkers of 
dietary intake18 and at once comprehensively characterises diet-related metabolic responses in human 
biofluids.19 Urinary biomarkers reflect the end product of metabolism. Urinary biomarkers combined with 

 
 
 
 
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assessment of compounds in food, blood (controlled to maintain homeostasis), and changes in gut 
microbial composition will provide a systematic approach to aid identification of underlying mechanisms of 
fruit and vegetable-induced BP effects. 
 
Using metabolomic strategies, small, short-term intervention studies have identified potential biomarkers 
for citrus fruits (proline betaine) and broccoli (S-methyl-L-cysteine sulfoxide (SMSCO)) in healthy 
participants.20,21 In a large-scale epidemiologic cohort study, the PI investigated their validity by combined 
analyses from high quality dietary data (24-hour dietary recalls), measured BP data and 24-hr urine 
collections measured by NMR spectroscopy from 2,032 US and 449 UK participants. In both populations 
and across clinic visits, consistent and strong correlations were found for intakes of citrus fruit with proline 
betaine (0.72), 4-hydroxyproline betaine (0.63) and several co-metabolites (0.22-0.49) related to the same 
metabolic pathway and for cruciferous vegetables with S-methyl-L-cysteine sulfoxide (SMSCO, 0.39). These 
biomarkers will be highly valuable as objective assessment of citrus fruits and cruciferous vegetable intake, 
as unbiased estimates of associations with disease outcomes in large-scale epidemiological studies, and as 
objective measures of compliance in intervention studies. 
 
Although meta-analyses of prospective cohort studies indicate that higher intakes of citrus fruits and 
cruciferous vegetables lower the risk of CVD8, limited evidence exists from intervention studies. Few small, 
acute interventions showed inconsistent findings on BP and endothelial function after supplementation of 
flavanones and/or orange juice25-27, while little evidence exist on cardioprotective effects of cruciferous 
vegetables. 28-30 Based on the evidence from prospective cohort studies and recent identification of strong, 
specific, and validated urinary markers for these metabolites, we will test increased intakes of citrus fruits 
and cruciferous vegetables on vascular function in this intervention study as proof of concept to develop 
future, larger intervention studies. We will not only test increased intakes of citrus fruit and cruciferous 
vegetables against low fruit and vegetable consumption, but also against increased intakes of other fruits 
and vegetables to rule out the effect of the higher amount consumed. 
 
In this pilot study, we will take into account the complexity of the whole food and will test the effects of 
higher intakes of citrus fruits and cruciferous vegetables on vascular function and cardiometabolic risk 
factors using predefined novel biomarkers to measure compliance. The simultaneous approach of 
measuring metabolites in various biofluids enables us to further validate these biomarkers and to assess 
changes with other urinary and circulatory metabolites to improve our understanding of potential 
underlying mechanisms and pathways. Additionally, we will also test cognitive function reported to relate 
to improved blood flow and neural activity following higher fruits and vegetable intakes. 
 
3. STUDY DESIGN 
 
This is a fully controlled randomised cross-over trial with three 14-day dietary intervention periods, 
preceded by one week run-in period and separated by one week wash-out periods, 9 weeks in total, in 36 
apparently healthy, untreated, prehypertensive participants aged between 40 and 65 years. 
 
3.1 Study objectives and outcome measures 
 
To assess the effect of types of plant foods on vascular function in untreated prehypertensive participants. 
 
Primary outcome 
 
Changes in systolic and diastolic blood pressure (mmHg) measured by automated oscillometric 
device (average of 3 measurements). 
 
Secondary outcomes 
 
Changes (end minus baseline values) in: 
1. Arterial stiffness assessed by pulse wave velocity and pulse wave analysis 
2. Cardiovascular risk factors measured in fasted plasma samples: 

 
 
 
 
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a. Lipid profiles: total cholesterol, high-density lipoprotein cholesterol, triacylglycerides (low-
density lipoprotein cholesterol will be calculated using the Friedewald formula) 
 
b. Insulin resistance: glucose, insulin, HbA1c (HOMA-IR will be calculated using fasting glucose 
and insulin values) 
3. Markers of endothelial function measured in fasted plasma samples (e.g. C-reactive protein) 
4. Markers of low-grade inflammation measured in fasted plasma samples (e.g. Interleukin-6) 
5. Urinary and circulatory metabolite profiles and individual metabolite levels 
6. Established objective urinary and circulatory markers of food intake: 
a. Fruits and vegetables: vitamin C, carotenoids, potassium, nitrate 
b. Salt: sodium 
c. Protein and muscle mass turnover: urea nitrogen and creatinine 
7. Composition of gut microbiota to be measured from stool samples 
 
8. Cognitive function measured by six computerised cognitive tests to assess four main cognitive 
domains: memory, processing speed, attention, and executive function 
9. Self-rated general health and mental well-being 
10. Average total, daytime, night time 24-hr ambulatory blood pressure (in subsample) 
11. Average objective physical activity intensity and sleep time (in subsample) 
 
 
3.2. Study methodology 
 
The study involves two screening moments (one screening questionnaire/interview to complete at home 
and one screening visit) followed by enrolment onto a 9-week randomized, placebo-controlled, cross-over 
intervention study. Each intervention period will be preceded by one week run-in period and separated by 
one week wash-out period in which participants will consume an average UK diet, low in plant foods. 
During the three 14-day intervention periods, participants will consume an average UK diet with increased 
amounts (8 servings per day) of specific types of fruits and vegetables (Table 1) or low in fruits and 
vegetables (control group, ≤2 servings per day). The diets will be fully controlled, provided by the 
researchers and delivered at the participants’ home once a week. Participants will be involved for 9 weeks 
in total and will be invited to attend the Cambridge Epidemiology & Clinical Trials Unit testing sites 7 times 
in total. 
 
Screening questionnaire/interview (15 minutes) 
Consent/Screening visit (clinic visit, 2 hours) 
Week 1: Run-in period 
Week 2-3: 14-day dietary intervention 
Week 4: Wash-out period 
 
Week 5-6: 14-day dietary intervention 
Week 7: Wash-out period 
Week 8-9: 14-day dietary intervention 
 
Screening questionnaire/interview (15 min) 
 
Respondents will be send by mail a screening questionnaire. Participants will be invited to complete and to 
return the screening questionnaire in the provided envelope to the researchers. If preferred, respondents 
can be contacted by telephone to assess suitability. The researchers will assess eligibility using the returned 
questionnaires and will contact all respondents by telephone to inform them if eligible. Eligible respondents 
will be sent the patient information sheet. Having read the information sheet, those who wish to continue 
the enrolment process will be invited to attend the Cambridge Epidemiology & Clinical Trials Unit testing 
sites for a consent visit and to continue screening. 
 
 

 
 
 
 
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Consent/screening visit (clinic, 2 hours) 
 
Potential participants will be asked to visit the Cambridge Epidemiology & Clinical Trials Unit testing sites. 
The respondent will receive detailed information of the study protocol and their eligibility before obtaining 
informed consent. Questions and concerns will be discussed with the researchers before giving formal 
consent. Potential participants will then be eligible to continue the screening process. 
 
Potential participants will be asked to complete a general health and lifestyle questionnaire. Automated 
office BP (twice with >30 min apart), anthropometrics and body composition will be measured. If eligibility 
criteria are met, the participant will be enrolled and randomly allocated to one of the intervention 
sequences. The participant will be asked to complete food diaries for 3 days (2 week and 1 weekend days) 
and a salt intake questionnaire to assess habitual dietary patterns and to return to the researcher by post 
for analyses. In the event of any abnormalities of test results, the participant will be informed and will be 
advised to discuss with their GP. 
 
3.3. Dietary intervention 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: Schematic representation of the study design and clinic visits 
 
The dietary interventions will be based on an average UK diet with similar macronutrient content: 
 
Control group: 1 portion of fruits plus 1 portion of vegetables, of commonly consumed types per day. 
Intakes are at 25th percentile of UK consumption (NDNS) and will exclude citrus fruits, cruciferous 
and allium vegetables. 
 
Treatment 1: 4 portions of fruits plus 4 portions of vegetables, of commonly consumed types per day 
excluding citrus fruits, cruciferous and allium vegetables. 
Treatment 2: 4 portions of citrus fruits plus 4 portions of cruciferous vegetables per day. 
 
The control group and treatment 1 will be provided with similar types of fruits and vegetables, but in 
different amounts, to investigate the importance of amount consumed (Table 1). Treatment 3 contains 
citrus fruits and cruciferous vegetables to test the importance of targeted types of fruits and vegetables 
consumed. Numbers of fruit and vegetable portions for the treatment groups are according to 
recommendations of the Dietary Approach to Stop Hypertension (DASH) diet.1 Fruits and vegetables will be 
provided as snack, salad, cooked in mixed dishes, as 100% juice, or as soup. Allium vegetables (onion, garlic, 
leek, etc) will be restricted throughout the study as these vegetables also contain SMSCO. Also any other 
fruit or vegetable containing food will be restricted e.g., jam and fruit cakes. The diets will be matched for 
servings of other food groups and macronutrient balance according to average UK habitual diet. Duplicates 
of daily provided diets will be collected to analyse average nutritional compositions of the diet. 
 
Meals and snacks covering 90% of daily energy needs will be delivered once a week at the participants’ 
home according to a weekly menu cycle for each intervention sequence and accompanied by daily menus. 
Each day, 10% of the calorie intake is free for the participant to choose from a list of food or beverage 
options (to be provided by participant). This may include a maximum of one daily unit of alcohol, equalling 
10 to 15 gram of ethanol. A run-in period (control diet) is to minimize the effect of habitual diet intake

 
 
 
 
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before start of intervention, washout period (control diet) precedes each intervention sequence to ensure 
that potential cardiometabolic effects of previous diet or treatment are eliminated. 
 
Table 1. Types of fruits and vegetables provided in each intervention sequence 
 
Control & Treatment 1 Treatment 2 
Common fruits 
Citrus fruits (Rutaceae) 
Apple 
Clementine 
Banana 
Grapefruit 
Grape 
Lemon 
Pear 
Lime 
Mandarin 
Orange 
Common vegetables 
Tangerine 
Carrot 
Cruciferous (Brassicaceae Oleracea) vegetables 
Cucumber 
Broccoli 
Sweet pepper 
Brussels sprouts 
Tomato 
Cabbages, including savoy, white and red 
Cauliflower 
Collard greens 
Kale 
Kohlrabi 
 
 
Diet control for weight maintenance is a crucial part throughout this study. Diets will be tailored to 
individuals’ energy requirements measured by bioelectrical impedance analysis and the validated Mifflin-St 
Jeor equation that estimates basal metabolic rate.31 Body weight and appetite profiles will be measured at 
start and end of each intervention sequence to check weight maintenance and feelings of hunger and 
satiation. Menu cycles at various caloric levels will be developed, and if needed, to match energy 
expenditure to maintain body weight. Participants will be asked to maintain usual lifestyle habits including 
physical activity levels throughout the study. Participants will be asked to complete a validated 
questionnaire at each visit to measure any changes in physical activity. The researchers will have weekly 
telephone contact with participants to discuss issues relative to the provided diet or if any lifestyle changes. 
 
Compliance to dietary intervention 
 
To aid compliance, all meals and snacks will be delivered on a weekly basis to participant’s home 
accompanied by instructions, daily menus and food checklists. The participant will be asked to complete 
the food checklists daily to report compliance to the intervention diet, the food choice for the remaining 
10% kcal, and any additional foods consumed not provided by the study. Participants will be asked to 
return the completed checklist and uneaten foods at the next clinic visit. Compliance will also be checked 
with objective established and novel dietary biomarkers. 
 
3.3. Study measurements 
 
Participants will be invited to attend the Cambridge Epidemiology & Clinical Trials Unit testing sites for 
clinical measurements at days 1 and 15 of each intervention sequence having fasted from 10 pm the 
previous day. Participants will be provided with containers and instructions for urine and stool sample 
collection. At each clinic visit, participant will bring 24 hour urine and a stool samples that were collected 
on the previous day, as well as completed food checklists and a questionnaire reporting any medications or 
dietary supplements used during the specimen collection. The participants will be reminded by text 
message to complete these collections and questionnaires. 
 
At arrival, a fasting venous blood sample will be taken with a volume that will not exceed 26 ml (156 ml for 
the whole study: see overview of fractions of blood samples in Table 3). Participants will be asked to void a 
spot urine for dipstick analyses to check pathological changes in the urine. Automated office BP, pulse wave 

 
 
 
 
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velocity, anthropometrics and body composition will be measured. Breakfast will be provided after 
completion of the measurements. Cognitive test will be completed at the laptop after breakfast which 
requires 12 to 15 minutes. We will use the same cognitive tests as currently used for the Airwave Health 
Monitoring study (MREC/13/NW/0588). 24-hr ambulatory blood pressure devices and AX3 acccelerometers 
became available at a later stage of the study. Therefore, these measurements will be conducted in a 
subsample of participants with main aim to validate the office blood pressure measurements and 
subjective physical activity questionnaire data. A subsample of the CIRCUS study participants will be invited 
to wear a 24-hr ambulatory blood pressure monitoring device at each visit and an accelerometer 
throughout the study period while performing their routine work and daily activities. The 24-hr ambulatory 
blood pressure monitoring device is worn around the waist which is connected with a wire to the cuff along 
the upper arm. The accelerometer is worn around the wrist as a watch. For the 24-hr ambulatory blood 
pressure measurement, participants will be trained to install the device themselves or will briefly attend 
the clinic the day before the actual clinic visit to have the device fitted by a trained researcher. After being 
notified by the device with a minimum inflation of the cuff, the device will automatically start 
measurement of blood pressure every 30/60 minutes for 24 hours. Participants who are willing to wear the 
accelerometer will be fitted or replaced the wrist band at each visit by a trained researcher. 
 
Table 2. Summary of assessments 
Screening 1 
Intervention period 1 
Intervention period 2 
Intervention period 3 
Assessment 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Total 
Informed consent 
X 
1x 
Screening questionnaire 
X 
1x 
Health & lifestyle questionnaire 
X 
1x 
Salt questionnaire 
X 
X 
2x 
Food diaries 
X 
3x 
Anthropometrics 
X 
X 
X 
X 
X 
X 
X 
7x 
Bioelectrical impedance 
X 
X 
X 
X 
X 
X 
X 
7x 
Blood pressure 
2x 
X 
X 
X 
X 
X 
X 
8x 
Pulse Wave Velocity 
X 
X 
X 
X 
X 
X 
6x 
Fasting blood sample 
X 
X 
X 
X 
X 
X 
6x 
24-hr urine 
X 
X 
X 
X 
X 
X 
6x 
Spot urine & dipstick test 
X 
X 
X 
X 
X 
X 
6x 
Faeces sample 
X 
X 
X 
X 
X 
X 
6x 
Cognitive tests 
X 
X 
X 
X 
X 
X 
6x 
Questionnaire including physical 
activity, dietary behaviour and 
X 
X 
X 
X 
X 
X 
6x 
appetite 
 
Table 3. Summary of blood samples 
 
Blood sample 
Quantity 
Vacutainer 
Purpose 
Serum Separator Tube (SST) 
6 mL 
Red 
Serum for biochemistry, metabolomics 
Serum Separator Tube (SST) 
5 mL 
Gold 
Serum for lipid profiles, hs-CRP, insulin 
Lithium Heparine 
6 mL 
Green 
Plasma for nutritional biochemistry (Vit C) 
Fluoride Oxalate 
2 mL 
Grey 
For glucose 
EDTA 
6 mL 
Purple 
Plasma and buffy coat for haematology and HbA1C 
Total blood sample 
25 mL 
 
 
At the end of the study, collected samples will be kept in locked freezer storage managed by the MRC 
Epidemiology Unit which hosts the Cambridge Epidemiology & Clinical Trials Unit.  
 
4. PARTICIPANT ENTRY 
 
4.1. Recruitment of participants 
 
We envisage that we will need to screen many people to find 36 eligible prehypertensive participants 
willing to participate in this study. We will therefore use various ways to recruit respondents: 
 

 
 
 
 
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 
by contacting the general pool of volunteers registered at the NIHR/Wellcome Clinical Research 
Facility and the Imperial College London, Division of Nutrition, and those registered at the 
Cambridge Epidemiology & Clinical Trials Unit of the MRC Epidemiology Unit, who participated in 
previous studies and who gave consent to re-contact

 
by advertisement, e.g. posters and online media, in staff rooms of wards, common areas, and 
waiting rooms of all the trust hospitals (Charing Cross, Hammersmith, and St Mary’s hospitals), and 
Addenbrookes Hospital in Cambridge, Prince of Wales Hospital in Ely, North Cambridgeshire 
Hospital in Wisbech  andImperial College London/University of Cambridge campuses – and via staff 
newsletters.

 
by contacting participants of the Airwave Health Monitoring Study who consented to be 
approached for recruitment of subsequent studies. The Airwave Study is an epidemiological

occupational cohort study of the police forces (officers and staff) in mainland Great Britain to 

investigate long-term health effects associated with use of the TETRA radio system.32 The Airwave 
Study obtained ethical approval from the National Health Service Multi-site Research Ethics 
Committee (MREC/13/NW/0588). Airwave participants who consented to be approached for 
recruitment to subsequent studies, who reside in London and Cambridgeshire, and who are 
identified eligible based on cardiometabolic risk factors measured at baseline will be approached. 

 
If necessary, we will extend our recruitment strategy to advertise at waiting rooms of local GP 
practises and/or direct recruitment via GPs.
 
When the study runs at the proposed new location, the Cambridge Epidemiology & Clinical Trials 
Unit, we aim to use the local Eastern CRN to recruit volunteers via searches of GP patient 
databases in Cambridge. Posters and adverts will be used if the GP surgeries does not create 
enough interest.
 
4.2 Pre-randomisation evaluations 
 
The screening phase to assess suitability for the study consists of a short screening questionnaire telephone 
interview and a consent/screening visit. Details of procedures are described in the Study methodology 
section and briefly presented here: 
 
1. Screening questionnaire/interview: Respondents will be invited to complete and return by mail 
the participant information sheet and a screening questionnaire to assess suitability. If preferred, 
the screening questionnaire can be done by telephone interview. Eligible participants who wish to 
continue the enrolment process will be invited to attend the Cambridge Epidemiology & Clinical 
Trials Unit testing sites for a consent/screening visit. 
 
2. Consent/screening visit: After receiving consent information and signing the informed consent the 
following assessments will be done to assess eligibility: general health and lifestyle questionnaire, 
automated office BP, and anthropometrics and body composition. The researchers will assess the 
results of both screening tests and will decide if the potential participant can enter the study. The 
participant will be asked to complete food diaries for 3 days (3 week and 1 weekend days) and a 
salt intake questionnaire in between visits to assess habitual dietary patterns. Eligible participants 
will be enrolled and randomly allocated to one of the intervention sequences. In the event of any 
abnormalities of test results, the participant will be informed and advised to discuss their results 
with their GP. 
 
Inclusion and exclusion criteria 
 
To limit potential confounding or misleading results, volunteers will be recruited according to inclusion and 
exclusion criteria as outlined in Table 4. 
 
 
4.2. Randomisation 
 
If eligible, the participant will be enrolled and randomly allocated to one of the intervention sequences. 
Randomisation will be carried out by an independent statistician.  
4.3. Withdrawal criteria 

 
 
 
 
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The safety of the study participants takes priority. Any significant adverse event (as assessed by the 
researchers) will halt the study and the ethics committee and sponsor will be informed as per standard 
protocol. All adverse events will be recorded and investigators will review each adverse event as it arises. In 
addition, participants will be free to withdraw at any time and are not required to give a reason. 
 
Table 4. Inclusion and exclusion criteria 
 
Inclusion criteria: 
Age 
40 to 65 years 
Automated office systolic BP 
Average systolic BP of 125-160 mmHg at 2 measurements seperated by >30 
min 
Antihypertensive medication use 
No use less than 3 weeks before screening 
Apparently healthy condition 
No reported current or previous diabetes mellitus, (secondary) 
hypertension, or metabolic, cardiovascular, renal, liver, thyroid, 
gastrointestinal diseases 
Body Mass Index 
20 to 35 kg/m2 
Smoking status 
Non-smoker (not smoked within last 6 months) 
Low habitual fruits and vegetable intake 
Average fruits and vegetables intake of <4 portions per day (UK average, 40- 
65 yr) or willing to reduce habitual daily fruit and vegetable intake ≤2 
weeks before enrollment 
Exclusion criteria: 
Use of other medications 
Any medications likely to interfere with energy metabolism, appetite 
regulation and hormonal balance, including inflammatory drugs or 
steroids, antibiotics, androgens, phenytoin, erythromycin or thyroid 
hormones and use of antibiotics. 
Alcohol intake 
> 21 units per week (females) or >28 units per week (male) and not willing 
to limit alcohol intake to maximum 1 unit/day ≤2 weeks before and during 
enrollment 
Physical activity 
≥10 h/week of moderate to vigorous physical activity 
Recent weight loss or gain 
≥3 kg in the preceding 3 months 
Use of dietary supplements 
Unwillingness to stop supplement use ≥2 weeks before enrollment and 
during intervention 
Other medical factors 
Pregnancy or lactation 
Intervention specific factors 
Unable or unwilling to consume provided diets during the intervention 
Unsufficient storage space for provided diets 
Food sensitivities or vegetarian/vegan diet by choice 
Participation in another intervention study at the same time 
Living >15 miles from the  Cambridge Epidemiology & Clinical Trials Unit 
testing sites, or not willing to travel more then 15 miles 
No signed informed consent 
 
5. ADVERSE EVENTS 
 
5.1. Definitions 
 
Adverse Event (AE): Any untoward medical occurrence in a patient or clinical study subject. 
Serious Adverse Event (SAE): Any untoward and unexpected medical occurrence that: 
 
results in death
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 
is life- threatening – refers to an event in which the subject was at risk of death at the time of 
the event; it does not refer to an event which hypothetically might have caused death if it was 
more severe.

 
requires hospitalisation, or prolongation of existing inpatients’ hospitalisation.
 
results in persistent or significant disability or incapacity
 
is a congenital abnormality or birth defect
 
Medical judgement should be exercised in deciding whether an AE is serious in other situations. Important AEs 
that are not immediately life threatening or do not result in death or hospitalisation but may jeopardise 
 
the subject or may require intervention to prevent one of the other outcomes listed in the definition 
above, should also be considered serious. 
 
5.2. Reporting procedures 
 
All adverse events should be reported. Depending on the nature of the event the reporting procedures 
below should be followed. Any questions concerning adverse event reporting should be directed to the 
Chief Investigator in the first instance. 
 
Non-serious AEs 
 
All such events, whether expected or not, should be recorded. 
 
Serious AEs (SEAs) 
 
An SAE form should be completed and faxed to the Chief Investigator within 24 h. However, relapse, death 
and hospitalisations for elective treatment of a pre-existing condition do not need reporting as SAEs. 
 
5.3. Referral procedures 
 
All SAEs should be reported to the Research Ethics Committee where in the opinion of the Chief 
Investigator the event was: 
 
 
‘related’, i.e. resulted from the administration of any of the research procedures; and
 
‘unexpected’, i.e. an event that 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 NRES SAE form. 
 
Local investigators should report any SAEs to the sponsor and their Local Research Ethics Committee and/ 
or Research and Development Office. 
 
 
Contact details for reporting SAEs, for attention of 
Dr Linda Oude Griep –  
FAX: 01223 769133 Tel: 01223 769220  
Email: Linda.OudeGriep@mrc-epid.cam.ac.uk 
(Mon to Fri 09.00- 17.00) 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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6. STATISTICS AND DATA ANALYSIS 
 
The statistical power calculation is based on BP-lowering effects (-7.6±1.4/-5.3±1.4 mmHg) of a DASH fruit 
and vegetable diet (+4.5 servings/day) vs average US diet in 15 obese hypertensive participants with an 
average BP of 136/90 mmHg in a 3-wk cross-over study with nutritional advice (no controlled diet).33 
Assuming a conservative detectable effect size (-1 mmHg SBP) considering differences in design and type of 
participants, 18 participants would be sufficient to detect a difference of 1 mmHg in SBP between groups 
(two-sided test at 5%, SD SBP change of 1.4 mmHg, 80% power). As we use block randomisation with 6 
intervention sequences, internal independent statisticians have advised to randomise a substantial number 
of participants per sequences. To further strengthen the study design, we will recruit and randomise 
(stratified by gender) 36 participants in total – with 6 participants in each group. 
 
Treatment effects will be calculated as the changes between values at start and end of each intervention 
sequence. Treatment effects will be tested by comparing changes of each of the increased fruit and 
vegetable interventions with changes during low fruit and vegetable period (control group), and of the 
citrus fruit and cruciferous vegetables period with increased commonly consumed fruit and vegetable 
intervention. Linear mixed models will be used for repeated measures to compare changes. Treatment and 
period will be set as fixed effects and subject as random effect. To test for carryover effect, the previous 
treatment will be included in the model. Statistical significance will be set at 2-sided P-value of 0.05. The 
researcher performing the statistical analyses will be blinded to the treatment groups. 
 
7. REGULATORY ISSUES 
 
ETHICS APPROVAL 
 
This study has been ethically approved by the London Brent Research Ethics Committee (17/LO/0862). The 
study will be conducted in accordance with the recommendations for physicians involved in research on 
human subjects adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions. 
Amendments to the protocol should be approved by the sponsor before being sent to ethics. After ethical 
approval, all amendments must have Trust R&D approval before they can be implemented. 
 
CONSENT 
 
Consent to enter the study must be sought from each participant only after a full explanation has been 
given, an information leaflet offered, and time allowed for consideration. Signed participant consent should 
be obtained. The right of the participant to refuse to participate without giving reasons must be respected. 
After the participant has entered the study the clinician remains free to give alternative treatment to that 
specified in the protocol at any stage if he/she feels it is in the participant’s best interest, but the reasons 
for doing so should be recorded. In such cases, the participants remain within the study for the purposes of 
follow-up and data analyses. All participants are free to withdraw at any time from the study without giving 
reasons and without prejudicing further treatment. 
 
CONFIDENTIALITY 
 
The Chief Investigator will preserve the confidentiality of participants in the study and is registered under 
the Data Protection Act. 
 
INDEMNITY 
The University of Cambridge holds adequate provision for insurance or indemnity to cover liabilities which 
may arise in relation to the design, management and conduct of the research project.  
 
SPONSOR 
 
The University of Cambridge will act as the main sponsor for this study.  
 
 

 
 
 
 
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FUNDING 
 
This research project is funded by the Academy of Medical Sciences. Travel expenses on public transport to 
and from the Cambridge Epidemiology & Clinical Trials Unit testing sites will be reimbursed up to a 
maximum of £20 per day for all visits (including screening visit) for both eligible and non-eligible volunteers. 
There will be no additional reimbursement during the intervention, however all meals and snacks during 
the 9 week intervention will be provided by the research team and delivered at participant’s home. 
 
AUDITS AND INSPECTIONS 
 
The study may be subject to inspection and audit by The University of Cambridge under their remit as 
sponsor and other regulatory bodies to ensure adherence to GCP and the NHS Research Governance 
Framework for Health and Social Care (2nd edition). 
 
8. STUDY MANAGEMENT 
 
The day to day management of the study will be co-ordinated by Dr Linda Oude Griep. 
 
9. PUBLICATION POLICY 
 
The findings of the research will be published in peer-reviewed journals and will be presented at targeted 
academic meetings to ensure wide reach of the results. In addition, we will be collaborating with patient 
groups and professional groups to disseminate the findings via multiple media channels such as patient 
association publications, print and broadcast media. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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