This randomized crossover trial (RCT) investigates the acute effect (over a 6-hour period) of a meal containing varying serving sizes (doses) of freeze-dried oyster mushroom powder, on the cognitive behaviour and markers of metabolism and inflammation related to neuronal health in healthy adults aged 60-80 years old. The study will involve a screening visit and four testing visits, with a week interval between each. During the four testing visits, cognitive-mood battery tests will be taken at baseline and then at 2-, 4- and 6-hour intervals following the consumption of the intervention meal. Also, a blood draw will be taken at the end of each testing visit day to allow the determination of inflammatory, metabolic and neuronal markers.

The Pleurotus oyster species is a common edible mushroom rich in ergothioneine, a bioactive compound with known neurocognitive benefits. The aim of the OYSACO RCT is to provide a clearer understanding of how a meal containing varying serving sizes (doses) of freeze-dried oyster mushroom powder, may acutely regulate cognitive improvements in humans during the immediate postprandial period. The investigators will investigate the neurological and physiological changes underlying these benefits.

During the OYSACO RCT, participants will attend the University of Reading, Psychology department on five separate occasions, with a week interval between each visit. Specifically, in the pre-screening phase, to check for eligibility, the participants interested in our study will be sent a link to REDCap containing online versions of a Health and Lifestyle Questionnaire containing basic demographic questions (eg age, sex, heigh, weight, nationality, disease history and medication use) and the Epic Norfolk Food Frequency Questionnaire (FFQ), to assess their habitual dietary intake. 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. Three drops of blood will be taken from the tip of the either the index or ring finger for this measure. 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. The order of intervention will be randomised, and participants will be asked to follow a low fibre and flavonoid diet for 48 hours in advance of testing. Before each test visit, the participants will be asked to fast for 12-hours and then to consume only a slice of toast with butter and 1 glass of water at 07:00 a.m., before attending the university. As soon as the participants arrive at our laboratory at 08:00 a.m., baseline cognitive and mood tests will be completed. After completing the baseline measures, the participants will receive one of the 3 mushroom interventions or the control intervention. The procedure on each test day will be identical except for the intervention meal. Specifically, the intervention meals will consist of a noodle soup containing the equivalent of 0.5, 1 and 2 servings of powdered Pleurotus oyster mushroom or a calorie-matched control non-mushroom noodle soup containing a mixture of maltodextrin and cornflour. The ingredients present in the intervention meals are all standard ingredients used in the food industry and are included at safe levels. Allergy information will be available on request. The dried oyster mushroom powder was prepared according to the Federal Good Manufacturing Practices and US Food Guidelines and is certified free from unauthorised pesticides, and microbiological and other contaminants.

Two-hours after consuming the intervention, the participants will be asked to complete the same cognitive and mood tests, and a calorie-matched lunch will then be provided between 12:00-12:30 p.m. containing a chicken sandwich, a packet of crisps and a glass of water. Participants will then be asked to complete the cognitive and mood tests a further two times (4-hours and 6-hours post-intervention) and a blood test will be drawn at the end of the visit.

In addition to the cognitive tests, palatability measures will be taken immediately after the consumption of the intervention meal while at the end of each cognitive test session (at baseline, 2-, 4- and 6-hours), ratings of subjective appetite and fullness will be recorded using an online analogue scale. At the end of the 5-week study, participants will be asked to complete a brief dietary questionnaire, consisting of specialised questions, relating to their habitual mushroom intake.

In terms of cognitive testing, there will be four cognitive task battery sessions taking place at baseline and then at 2-, 4-and 6-hours post-intervention, to assess participants' episodic and working memory, psychomotor and executive function and mood domains. Each battery will be matched for difficulty and order of presentation will be randomised. Although the exact cognitive battery is yet to be confirmed, all tasks will be completed on the computer and should take no longer than 45 minutes to complete. The battery is likely to contain the following or similar tasks:

• Positive and Negative Affect Schedule (PANAS-SF)- In this mood task, participants are asked to rate their emotion on a 5-point Likert scale, in response to 20 mood related adjectives (10 relating to positive emotions and 10 to negative emotions).
• Rey Auditory Verbal Learning Task (RAVLT)- This episodic memory task consists of 5 consecutive free recalls of the same 15 nouns presented as an auditory list (list A), followed by recall of a further 15 nouns presented as an interference list (list B) which is recalled only once. List A is subsequently recalled straight after list B and then again after a longer delay of around 30 minutes.
• Corsi block tapping task (CBTT)- In this spatial working memory task, participants are shown with 9 arranged blocks positioned on a computer screen and are asked to reproduce given sequence by clicking on the blocks (using the mouse) in the same sequence as they see them.
• Simple and complex finger tapping task- In this motor function task, participants tap on a key as quickly as possible with the index finger of their dominant hand for 1 minute. They then tap out a specific sequence using 4 fingers, again for 1 minute.
• Task Switching Task (TST) - In this executive function task, participants view 8 spaced radii of a circle above and below a bold line and a stimulus digit selected from 1-9 (except 5) appears clockwise in each segment, either above or below the bold line. Depending on the stimulus position in the segments, participants perform different tasks with each being switched in every 4 trials.
• RAVLT word recall- After a period of time, participants are asked to recall as many words as possible from list A.
• RAVLT word recognition- In this delayed memory task, participants are shown a sequential list of 50 nouns containing the words from lists A and B from the previously described RAVLT task plus 20 additional words not previously heard, and are asked to indicate those from list A.

During each test day, participants will have one 9-millilitre blood draw taken from their arm, 6-hours after receiving the intervention meal. Following each draw, the serum will be separated via centrifuge and stored at at -80°C until analysis is complete. Whole blood samples will not be stored at any point during the study. Blood serum will be analysed for anti-inflammatory and metabolic ability by measuring the levels of fasting glucose and lipids in the blood, the markers of inflammation, as well as the levels of the brain derived neurotrophic factor (BDNF), a signalling protein known to be related to neuronal signalling and memory function.

Inclusion Criteria:

• Aged between 60-80 years old
• Have normal vision and hearing
• Have healthy status
• Have a BMI<30

Exclusion Criteria:

• Smokers or those consuming more than the recommended units of alcohol per week
• Vegetarians/ Vegans
• Being diagnosed with any psychiatric or neurologic conditions (e.g. schizophrenia, depression, cognitive dementia)
• Being diagnosed with cardiometabolic disease (including type II diabetes and cardiovascular disease), or suffer from hypertension or thrombosis related disorders
• Being anaemic
• Currently taking regular vitamin supplements or disease medication such as anticoagulant or antiplatelet medication and antidepressants etc
• Diagnosed with an eating disorder
• Be allergic to mushrooms, or any other nutrients present in the intervention meals

All personally related collected information, including the paper consent forms, will be kept in locked filing cabinets in the Nutritional Unit of the School of Psychology. The digital health questionnaires used for the purpose of pre-screening participants as well as any digital research data will be stored in the Research Electronic Data Capture (REDCap) database, stored on the University secure network cloud (UREC).

All relevant study outcomes described in the Ethics Review Application Form (including the demographic and anthropometric data (such as age, sex, BMI, blood pressure) as well as the performance scores of the battery tests assessing cognitive function and mood and the results of the blood tests measuring immunological and metabolic function) will be preserved and made available in anonymised form, so that they can be consulted and re-used by others.