Experimental design

In order to answer these research questions a series of experiments were designed:

 Experiment 1: Piloting of cognitive tasks and an examination of the effects of repeated cognitive testing on task performance (Chapter 3).

 Experiment 2: A dose-response study of cognitive and blood pressure changes following acute anthocyanin-rich blueberry supplementation in healthy young adults (Chapter 4).

 Experiment 3: A dose-response study of glycaemic response following acute anthocyanin- rich blueberry supplementation in healthy young adults (Chapter 5).

 Experiment 4: A dose-response study of cognitive and blood glucose changes following acute anthocyanin-rich blueberry supplementation in healthy young adults (Chapter 6).

The majority of intervention studies reviewed in the previous chapter opted for a crossover design. In this type of design, a single group of participants take part in each experimental condition of the study. Participants effectively act as their own controls, thereby minimising the impact of between- subjects variance and so improving statistical power. Similarly here, a crossover design was

implemented for each blueberry intervention study. As cognitive practice effects are known to be an issue in experimental designs with a repeated testing component, the magnitude of these effects was fully investigated during the piloting of the cognitive tasks and appropriate methodology was implemented for reducing their impact (discussed in detail in Chapter 3). Methodological limitations, identified in several of the reviewed intervention studies, were their lack of an appropriate control condition or an omission of baseline testing. In each of the three intervention studies described in this thesis (Chapters 4, 5 & 6), appropriate matched control conditions were included alongside the

28 blueberry interventions. Baseline measurements were recorded at each test visit before

interventions were administered in order to control for random variations in performance across multiple test visits. In accordance with a further standard of good practice in intervention studies, all testing was performed double-blind in order to prevent potential experimenter effects or demand characteristics from influencing participant performance.

2.1.1 Power analysis

A priori power analysis was performed using GPower 3.1 to determine the minimum number of participants required for each experiment in order to achieve a statistical power of 0.8 with an alpha level of 0.05 (Cohen, 1992). For Experiment 1, assuming an effect size of d=0.47 (Bartels, Wegrzyn, Wiedl, Ackermann, & Ehrenreich, 2010; Donovan & Radosevich, 1999), 30 participants were determined as sufficient to detect an increase in cognitive performance between repeat test

sessions. For Experiments 2 & 4, assuming an effect size of d=0.45 (Bell, Lamport, Butler, & Williams, 2015), 32 participants were sufficient to detect an increase in cognitive performance between the control and blueberry conditions. For Experiment 3, assuming an effect size of d=0.71 (Törrönen et al., 2010), 14 participants were deemed sufficient to detect a reduction in peak glycaemic response between the control and blueberry conditions. Extra participants were recruited where possible to allow for attrition/drop outs throughout the course of testing.

2.2 Ethical approval

All studies were approved for ethical conduct by either the School of Psychology Research Ethics Committee (SREC), or the University of Reading Research Ethics Committee (UREC), in accordance with University of Reading guidelines. Evidence of ethical approval for all studies can be found in Appendix A.

2.3 Participants

2.3.1 Recruitment

Participants were healthy young adults, aged 18 to 40, recruited from staff and student populations at the University of Reading. This population was selected as data outlining bioavailability

(Rodriguez-Mateos, Feliciano, Cifuentes-Gomez, & Spencer, 2016) and vasoreactivity (Rodriguez- Mateos et al., 2013) following acute doses of blueberry anthocyanins have already been published for this age group. Similarly, the literature review in Chapter 1, identified cognitive and mood effects in young adults following blueberry intervention. Recruitment was carried out via postings to group

29 email listings, campus notice boards, and the Psychology Department Undergraduate Research Panel.

2.3.2 Screening

Aside from smoking status, no screening restrictions were in place for Experiment 1. However, all participants were screened for suitability before inclusion in any of the blueberry intervention studies. Participants were required to be non-smokers, free from pregnancy, food allergies, diabetes mellitus (Type 1 & Type 2), and any conditions requiring hypotensive or anticoagulant medication. Full eligibility criteria are reported in Appendix B. A copy of the health questionnaire used for screening can be found in Appendix C.

2.3.3 Participant demographic data

Health and lifestyle demographic data comparing participants for all experiments are shown in Table 2.1. The participant profile remained consistent between all experiments.

2.3.4 Informed consent

Before participation in any of the experiments reported here, prospective participants were emailed an information sheet providing detailed information about the study. If after reading the information participants were happy to continue, then they were required to sign a consent form at the

beginning of their first test visit. Information sheets for all experiments can be found in Appendix D. Consent forms for all experiments can be found in Appendix E.

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Table 2.1 Participant demographic data

Demographic Experiment 1.1 1.2 2 3 4 N 29 33 45 17 41 Gender male 9 3 12 4 11 female 20 30 33 13 30 Age(years) Mean 25.6 20.8 20.9 24.1 23.5 SD 7.7 4.9 3.6 4.9 5.1 BMI (kg/m2) Mean N/R N/R N/R 23.8 23.4 SD 3.6 5.0

Fruit & veg (daily portions) Mean N/R N/R 4.1 5.3 4.4

SD 1.4 2.3 1.9

Tea & coffee (daily cups) Mean N/R N/R 1.7 2.2 1.3

SD 1.8 1.5 1.2

Alcohol (weekly units) Mean N/R N/R 5.1 3.6 3.3

SD 6.8 5.0 5.0

Exercise (weekly hours) Mean N/R N/R 2.9 3.0 3.3

SD 3.3 2.3 3.5

N/R Not recorded

2.3.5 Low polyphenol diet

Before participation in any of the blueberry intervention experiments reported here, participants were required to follow a low polyphenol diet for the 24 hours prior to each test visit. Similar methodology has been employed by studies investigating bioavailability (Rodriguez-Mateos et al., 2016), vasodilatory response (Rodriguez-Mateos et al., 2013), cognition (Dodd, 2012), and mood (Khalid et al., 2017) following acute wild blueberry intervention. In addition to food and supplement restrictions, participants were asked to abstain from alcohol, fruit juices, and caffeine containing beverages such as energy drinks, cola, tea, coffee or cocoa. Participants were also asked to fast (no food or drink) for 2 hours immediately prior to attending each test visit. Participants were asked to

31 eat the same breakfast before each test visit and to ensure that breakfast was consumed before the 2 hour fast, with baseline cognitive testing commencing at 9am. This methodology was introduced in order to minimise the interfering effects of habitual dietary polyphenols on each of the measured outcomes following blueberry intervention, and to minimise the impact of dietary changes on test performance between visits. The 2 hour fast ensured that participants were thirsty enough to consume the intervention beverage, whilst minimising the confounding effects of any breakfast food intake. A copy of the dietary requirements can be found in Appendix F. At each test visit participants retrospectively documented their 24 hour food intake using the record sheet found in Appendix G. Minor transgressions were noted and, where necessary, participants were asked to modify their pre- test diet.

2.3.6 Payment

Participant payments were standardised in line with current University of Reading guidelines. Psychology undergraduates received 1 course credit per hour of testing completed. All other participants received expenses payments of £5 per visit.