The Effects of a Single Dose of Wild Blueberries on Mood and Cognition in Healthy Young Adults: a Randomized, Double-blind, Placebo-controlled, Crossover Study
Phase: NA
Conditions: Mood, Affect, Cognition
Sponsor: University of Reading

 
 
 
 
 
Study Protocol and Statistical Analysis Plan 
Study title: The Effects of a Single Dose of Wild Blueberries on Mood and Cognition in Healthy 
Young Adults: a Randomized, Double-blind, Placebo-controlled, Crossover Study 
Date: 27 May 2021 
 
 
 
 
 
 
 
 
 

1. Sample Size  
A previous study with a similar crossover design found a significant effect of a blueberry drink 
intervention on mood using a sample size of 21 (Khalid et al. 2017). For the purpose of this study, 
we decided to include 30 participants – 15 male and 15 female, to be able to study the influence 
that sex might have on how the intervention affects our outcomes of interest.  
2. Recruitment and Screening  
Participants will be recruited using opportunity sampling. The study will be advertised online, 
through email distribution, and with posters across the University of Reading and the local 
community. Before interested participants are enrolled in the trial, we will invite them to the lab 
for a screening and familiarization session to determine if they meet the eligibility criteria, conduct 
a detailed baseline assessment of predictive factors, and give them an opportunity to become 
familiar with the cognitive task battery so as to reduce the influence of practice effects. Participants 
will be compensated with £100 ($141) for completing the study. 
3. Counterbalancing and Blinding Procedures  
The participants will receive both interventions in a counterbalanced manner, i.e. half will be 
allocated to the order “blubbery  placebo” and the other half will receive the placebo first. The 
study will be double-blind. A researcher not involved in recruitment or data collection will prepare 
the intervention drinks and administer them in opaque shaker bottles. Thus, both participants and 
investigators will be blinded to the treatment assignment throughout the study.  
4. Testing Protocol  
Each participant will undergo two identical testing sessions at the Nutritional Psychology Lab. On 
testing days, participants will arrive at the lab after an overnight fast and will consume a standard 
breakfast of oat porridge. They will have been asked to follow a low-flavonoid diet on the day 
prior to testing. Next, participants will provide an intravenous blood sample and will complete the 
baseline cognitive task battery. When finished, they will consume the intervention drink they have 
been allocated to, prepared by mixing the blueberry or placebo powder with 250 ml water. 
Allowing 100 min for absorption, we will take another blood sample and will have participants 
repeat a different version of the same task battery.  

5. Statistical Analysis Plan   
The present study aims to investigate the acute (2 hours) effects of a wild blueberry intervention 
on mood and cognition in a population of healthy young adults. On the recommendation of a 
statistician, all repeated measures data will be analyzed in RStudio using linear mixed-effects 
models (LMMs). This technique can be used to model variance relating to both fixed parameters 
such as experimental doses and random parameters such as individual differences between 
subjects, within multiple layers of the same model. We will adjust the model for baseline variables, 
such as sex, fruit and vegetable consumption, physical activity level, alcohol use, and systemic 
inflammation, where the inclusion of these covariates improves the fit of the model. Subjects will 
be included as a random factor as a way of controlling for non-independence of data from the same 
subjects. Where possible, an unstructured covariance matrix will be specified for the repeated 
observations on a subject within the same period. As LMMs do not require balanced data, subjects 
with missing data points will not be excluded from the analyses.  
 
References 
Khalid S, Barfoot KL, May G, Lamport DJ, Reynolds SA, Williams CM. Effects of Acute 
Blueberry Flavonoids on Mood in Children and Young Adults. Nutrients. 2017 Feb 20;9(2):158. 
doi: 10.3390/nu9020158.