16 Weeks' Dietary Supplementation With Iron and Iron + Vitamin C on Cerebral Blood Flow and Energy Expenditure in Women of Reproductive Age

Each participant will be required to attend the laboratory on four occasions. The first is comprised of a screening/training visit, which will take place in the afternoon for approximately 2.5 hours. This will also be between days 21-28/ the week before onset of their menstrual cycle. If they do not have menstrual bleeds then this appointment can be any afternoon. This will comprise: briefing of requirements of the study; obtaining of informed consent; confirmation of eligibility to take part, including collection of demographic data and health screening, and training on the cognitive and mood measures. The training session will follow standard operating procedures to decrease the chance of learning effects during main trials. Extra training will be given where necessary.

Participants will be required to complete questionnaires based upon food frequency, caffeine consumption and to estimate the number of hours of exercise completed on a weekly basis. A menstrual cycle questionnaire will also be completed to estimate menstrual blood loss and so that participants attend their testing visit during days 7-14 of their cycle. If participants do not have a menstrual cycle due to contraceptive methods, then appointments will be approximately two weeks apart. A finger-prick and venous blood sample will be collected from participants, which will be analysed for iron status. Those whose haemoglobin levels are <120g/L will be advised to seek advice from their GP. Participants who fell into the iron status category of iron sufficient (haemoglobin ≥120 g/L and serum ferritin > 20 µg/L) or non-anaemic iron deficient (haemoglobin ≥120 g/L and serum ferritin ≤ 20 µg/L) will be informed of their eligibility for the intervention study by email or phone between the initial visit and their next. This will include the option to enrol in an additional assessment of neuroimaging and energy metabolism.

For the testing visit, participants will be asked to fast for 12 hours prior to the visit, avoiding intake of all food and drink with the exception of water. They will also be asked to avoid alcohol and refrain from intake of 'over the counter' medication for 24 hours. Participants will arrive at the laboratory at a designated time in the morning. The following procedures will take place prior to cognitive and mood testing:

Review of continued conformity to eligibility criteria Adverse event and concomitant medication assessment Ensure that participant is in good health

Participants will then complete the baseline cognitive and mood assessments. Following this, participants are informed of their iron status and will be briefed regarding the requirements of the intervention study and additional assessment. Following this, informed consent will be obtained prior to starting the additional assessment. Participants will have a frequency domain near-infrared spectroscopy headband attached to the forehead to measure cerebral blood flow at rest for five minutes. Following this, participants will instead have a continuous wave near-infrared spectroscopy headband attached to the forehead to measure cerebral blood flow, and a face mask fitted to measure expired air. Participants will have another five minute rest before completing an additional battery of cognitive tasks for approximately 35-40 minutes. Once complete, participants will be randomised to one of three treatments and be provided with a treatment diary to log treatment consumption and any adverse events experienced.

Participants will return to the laboratory after 8 weeks to exchange treatment bottles and diaries in order to check compliance.

Participants will finally return to the laboratory after a further 8 weeks (16 weeks total). The same procedures will take place prior to cognitive and mood testing as completed at the baseline testing visit, with the addition of finger prick and venous blood sample and weight measurement. Participants will then complete the same cognitive, mood and neuroimaging and energy metabolism assessments as completed during the baseline testing visit.

Inclusion criteria

• Healthy
• Female
• Aged 18-49 (inclusive)
• Have a BMI of between 18.5-40
• Have an English bank account (required for payment)

Exclusion criteria

• Aged under 18 or above 49 years
• BMI lower than 18.5 or higher than 40
• Pre-existing medical condition/illness with some exceptions - please check with researcher
• Blood disorders (including anaemia) or any known active infections
• Current or past breast cancer diagnosis and/or mastectomy
• Smoking or use of any nicotine replacement products e.g. vaping, gum, patches
• Pregnant, trying to get pregnant or breast feeding
• Currently taking any prescription medication with some exceptions - please check with researcher
• Food allergies/sensitivities relevant to the study
• Regular use of dietary/herbal supplements within the last month (defined as more than 3 consecutive days or 4 days in total)
• Use of iron supplements within the past 4 months
• Have donated more than 300ml of blood in the past 3 months
• Have haemoglobin levels below 120g/L
• History of significant head trauma or suffer from frequent migraines that require medication (more than or equal to one per month)
• Learning difficulties, dyslexia, or colour blindness
• Visual impairment that cannot be corrected with glasses or contact lenses
• Currently taking part in any other clinical or nutritional intervention studies or have in the past 4 weeks
• Any health condition that would prevent fulfilment of the study requirements