A Randomized Controlled Trial to Investigate the Neurological, Inflammatory and Metabolic Effects of Acute Mushroom Intervention in Older Adults (OYSACO)
Phase: NA
Conditions: Cognitive Change, Mood, Metabolism, Inflammatory Response, Satiety
Sponsor: University of Reading

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STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
Study title: A Randomized Controlled Trial to Investigate the Neurological, Inflammatory and 
Metabolic Effects of Acute Mushroom Intervention in Older Adults.   
Acronym: OYSACO 
Date: 15 August 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Sample Size Calculation 
A power calculation based on a study with a similar design investigating the acute 
benefits of bioactive rich interventions on cognitive function in participants aged 60-80 
years, suggests that 30 participants should give sufficient statistical power (Bell and 
Williams, 2019). To allow for a 10% attrition rate, 33 older adults has been agreed to be 
recruited. 
2. Recruitment and Screening 
Participants will be initially recruited using opportunity sampling. The study will be 
advertised via email and posters will be placed around public areas (eg university area, 
sports hall, library, churches, GP practices). Furthermore, participants will be recruited 
from the Age UK Berkshire and the local community and if needed from the university of 
Reading research ageing panel.  
Before interested participants are enrolled in our study, they will be invited to attend a 
screening and familiarization session to our unit, to determine if they meet the eligibility 
criteria and they will be given an opportunity to become familiar with the cognitive task 
battery to reduce the influence of practice effects. Participants will be compensated with 
£200 for completing all study visits. 
3. Randomisation Process 
The study will be double blinded since neither the participants nor the researcher will 
know which intervention meal participants are receiving until the study is over. To 
achieve this, the Latin Square design (4X4) will be followed, by using blocked 
randomisation with random permuted blocks of four. Another individual not being 
involved in this study, will assign a code to each of the four intervention meals and these 
will be kept in a sealed envelope, concealed to the researcher enrolling and assessing 
participants. On the testing days, the intervention meal soups will be prepared by research 
personnel not involved in the conduct of the trial.  
4. OYSACO study Protocol 
During this study, participants will attend the University of Reading, Psychology 
department on five separate occasions, with a week interval between each visit.  
In the pre-screening phase, the participants interested in our study will be sent a link to 
REDCap containing online versions of a Health and Lifestyle Questionnaire and the Epic 
Norfolk Food Frequency Questionnaire (FFQ). Then, participants, will be contacted to 
attend a 2-hour screening session at our department, during which the participant’s 
weight, height and blood pressure will be checked, along with a finger-prick, to ensure 
that the participants are not anaemic. Furthermore, participants will complete the Raven’s 
Progressive Matrices (RPM) measure of fluid intelligence and will perform the cognitive 
battery tasks twice to control for practice effects in the run up to the test session days. 
One week after the screening visit, each participant will attend a further four test sessions, 
each separated by a week, where they will receive a different dose of freeze-dried oyster 
mushrooms or a control soup. Participants will be asked to follow a low fibre and 
flavonoid diet for 48 hours in advance of testing and these four testing days participants 
have to remain to our unit from 08:00 a.m.- 16:00 p.m. Figure 1 summarises the 
OYSACO testing protocol.  
 
 
 

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Figure 1: Study design of the 4-arm OYSACO RCT, investigating the dose-response effects of an 
acute diet containing ergothioneine-rich Pleurotus mushrooms on the cognitive, metabolic and 
inflammatory function in healthy older adults.   
(*) For the soup interventions, the Pleurotus mushrooms will be in a powdered form at varying 
doses (equivalent to 0.5 (soup 1), 1 (soup 2) and 2 (soup 3) mushroom servings (with 1 serving~8g 
powdered mushrooms). 
 
5. Statistical Plan 
The present study aims to investigate the beneficial effects of a meal containing Pleurotus 
oyster mushrooms on neurocognitive performance (objective I) and improvements in the 
blood serum measures of inflammation and metabolism (objective II) in the acute 
postprandial period (6-hours post-consumption) in older adults aged 60-80 years old.  
Participant sample characteristics will be tabulated using descriptive statistics, using 
SPSS software. Parametric tests will be used throughout having first confirmed that the 
test data meet the required assumption of normality. Statistical analyses of the cognitive 
measures will be performed using SPSS linear mixed modelling (LMM), employing an 
unstructured covariance matrix to model successive repeat measurements. LMM will be 
applied to model variance relating to both fixed parameters (eg experimental meal) and 
random parameters such as differences between participants. The analysis will involve the 
creation of both unadjusted models and adjusted models by controlling for baseline 
variables such as sex, fruit and vegetable consumption, physical activity level and health 
status. Baseline performance will be included as a repeated covariate. Subjects will be 
included as a random factor.  
In the primary analysis of the data in each cognitive task, the Visit Order (Visit), Time 
after receiving the experimental meal (Time), experimental meal (Intervention) and Time 
X Intervention interaction will be included as fixed factors in the model. A composite 
score will be also calculated by the summation of the z scores obtained from each 
cognitive task, to examine the effect of the intervention meal on the overall cognitive 
performance.  
The data obtained from the metabolic and inflammatory markers will be checked for 
normality before using parametric statistical tests. The blood serum markers obtained 
                         
               
               
               
             
                
              
                 
           
         
          
          
          
          
          
          
          
          
               
 
 
 
 
 
         
 
          
 
                      
 
 
 
 
                 
 
     
 
                    
 

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following the intake of the control meal will be compared with the blood serum markers 
obtained following the intake of the three mushroom containing soups. The SPSS LMM 
method will be employed, and the data analysis will be similar to the one performed on 
the cognitive data without the ‘Time’ being a fixed factor in the model, since the blood 
sample will be collected at one time point (6 hours post-intervention). Finally, a 
correlation will be made for the blood biomarkers and cognitive test scores at the 6-hour 
post-intervention time point.  
 
References 
Bell, L., Williams, C.M. (2019). A pilot dose–response study of the acute effects of 
haskap berry extract (Lonicera caerulea L.) on cognition, mood, and blood pressure in 
older adults. Eur J Nutr 58, 3325–3334. https://doi.org/10.1007/s00394-018-1877-9