Caffeine, DNA and Cognition

A double-blind, placebo-controlled crossover design that incorporates alternating periods of 'long-term' caffeine exposure and abstinence will be employed to investigate the inter-individual effects of caffeine on cognitive performance. The procedure that participants need to follow on each of the four weeks of the study is described in detail below.

1. Baseline visit: baseline questionnaire, anthropometry, familiarisation with study procedures and tasks. Participants will be supplied with week 1 supplementation (n= 18 capsules) and saliva vials (n= 6) for saliva sample collection. Participants will also be provided with decaffeinated coffee, tea and cocoa, depending on their preferences.
2. Days 1-6: placebo or caffeine capsules x 3 per day at 09.00, 11.00, and 15.00. Compliance with the caffeine and placebo supplementation will be assessed by a saliva sample at 17.00 every day for each of the days on long-term supplementation. Caffeine concentrations in blood and saliva correlate highly, and levels typically increase progressively during the day (due to intermittent consumption of caffeine beverages). The best single-sample bioassay of daily caffeine intake is provided by samples obtained in the late afternoon (James, 1994; James \& Rogers, 2005).
3. Days 5 and 6: In preparation for the 'challenge' days and to control for environmental factors which have been shown to affect inducibility of CYP1A2 enzyme, participants will be asked to abstain from alcohol for 48 h and avoid strenuous exercise for 24 h before the trials and avoid consumption of cruciferous foods (broccoli and Brussels sprouts) (Gunes \& Dahl, 2008). Finally, participants will be provided with diet diaries to record all food and beverages consumed during the 24 h before all four experimental trials.
4. Day 7 (Experimental day): Participants will be asked to present to the laboratory after an overnight fast and they will follow the procedure shown below:

09:00: Pre-supplementation saliva sample \& cognitive tasks

09:30: Supplementation with 3 mg / kg body mass caffeine or placebo

10:30: First post-supplementation saliva sample \& cognitive tasks

12:30: Second post-supplementation saliva sample \& cognitive tasks

13:00: A meal containing of fruit juice or soft drink and a cheese sandwich

15:30: Third post-supplementation saliva sample \& cognitive tasks

Saliva samples will be taken upon arrival and 1, 3 and 6 h post-supplementation. The first sample will be taken to verify adherence to the requirement not to take their morning supplementation, thus be in caffeine abstinence, regardless of the study arm. It has been shown that 1 mg/kg of caffeine results in caffeine plasma levels of about 1 μg/ml and salivary caffeine concentration may be expected to be about 70% of plasma levels (Walther et al., 1983). Mean saliva caffeine levels below 1 μg / ml have been previously reported for overnight caffeine abstinence (Evans \& Griffiths, 1999). Following caffeinated treatment, salivary caffeine levels will aid in determination of caffeine metabolism (Dodd et al., 2015).

The supplementation will be swallowed with water proportional to participant weight (3-ml/kg body mass). During the experimental days, participants will be asked to consume water ad libitum in their first trial and an equal volume in the subsequent trials. They will also be asked to avoid exercise between the trials.

The cognitive tasks will be performed on four time points: pre- and 1, 3 and 6 h post-supplementation. When using anhydrous caffeine in capsules, peak caffeine concentration usually occurs around 1 h post ingestion (Graham, 2001). Therefore, measuring caffeine metabolites for 1 h post-caffeine ingestion would mostly measure caffeine absorption and not metabolism, which is determined by CYP1A2 enzyme. Thus, studies investigating the effects of CYP1A2 genotypes on caffeine effects should focus on using protocols which last >1 h, since these effects may be more evident in events lasting longer than 1 h, where the metabolism of caffeine may have a more pronounced effect. It should also be noted that the half-life of caffeine is on average 4-6 h (Nehlig, 2018) in most adults and it is not yet known to what degree caffeine metabolism is altered between fast and slow metabolisers. Therefore, it is unknown at what time point there would be a large enough difference in the circulating levels of caffeine between fast and slow metabolisers to have a significant impact on the ergogenicity of caffeine (Southward et al., 2018). For this reason, we selected 3 different time points post-supplementation to complete the cognitive tasks: 1 h post-supplementation was selected to permit comparison with several previous caffeine studies, because it is the most frequently used time point to test cognitive performance post-supplementation in the literature (Carswell et al., 2020). We also added the 3 h and 6 h post-supplementation time points since they are within the average range of caffeine half-life and to investigate the differences in performance between 'fast' and 'slow' metabolisers. We hypothesise that those who metabolise caffeine faster would not maintain high saliva levels of caffeine throughout the 6 h event compared to those with a slower metabolism of caffeine.

At the end of each experimental day, participants will be supplied with the capsules for the following week, as well as the vials for saliva caffeine sampling.

Once provided, samples will be kept frozen at -20 °C until analysis. Salivary caffeine levels will be measured using the Enzyme Multiplied Immunoassay Technique (EMIT) by spectrophotometric method. The EMIT assay is a homogenous enzyme immunoassay intended for use in determining caffeine as a metabolite and will be preferred given that it is less invasive than using serum caffeine and has been shown reliable and reproducible (Tripathi et al., 2015). Serum caffeine levels greater than 30 μg / ml require dilution when analysed by EMIT assay.