Background.

The following socio-demographic variables will be collected for all participants regardless of veteran status: age, biological sex at birth, identified gender, sexual orientation, ethnicity, years and level of education; household income, occupational history. The “LGBT ban” in the UK military was officially lifted in 2000, following the implementation of the European Convention on Human Rights, which led to changes in UK legislation to protect the rights of LGBT individuals, including their right to serve in the military without fear of discrimination based on sexual orientation or gender identity. Prior to this, there were policies in place that prohibited lesbian, gay, bisexual, and transgender (LGBT) individuals from serving openly in the armed forces. As we are interested in lifetime and occupational stressors, such as bullying, harassment and discrimination (see below), we will ask participants for their identified gender and sexual orientation, as well as ethnicity, to understand whether these variables influence outcomes relating to psychosocial or other outcomes. Participants are reminded during the interview that they do not have to disclose information regarding these protected characteristics if they do not feel comfortable to do so and such refusal will not affect their participation in the study.

For military veterans, military-related information will be collected: serving status; length of service; service branch/unit; rank; number of deployments and their geographic regions; engagement type (regular, reserve), and combat role, if any.

General health.

All participants will complete a general health questionnaire that includes self-reported items such as: height, weight, history of past and current medical conditions, including neurodevelopmental conditions (such as Attention Deficit/Hyperactivity Disorder); current and past medications. All participants will be asked about history of Traumatic Brain injury (number of injuries, age at injury/ies); military veterans will be further prompted to report if their injury/ies were related to military service.

Endocrinological and reproductive health.

Female participants complete questions relating to endocrinological health and fertility, such as menopausal status, menopausal and menstrual symptoms, hormonal contraception and other hormonal use (e.g. hormonal replacement therapy), history of reproductive surgeries (e.g. hysterectomy); age of menarche and menopause (if applicable), history of pregnancies and pregnancy-related health conditions (e.g. gestational diabetes, hyperemesis gravidarum) and sexual health conditions affecting fertility. Male participants will complete questions relating to endocrinological health, such as hormone use, number of pregnancies fathered (both live and non-live births) as well as history of reproductive surgeries and sexual health conditions affecting fertility.

All participants, regardless of veteran status, will complete the following neuropsychological and psychosocial, biological and neurophysiological assessments:

Neuropsychological and psychosocial assessments.

The following measures will be employed to assess cognitive function.

The Repeatable Battery for Assessment of Neuropsychological Status [RBANS, 22] which includes 12 subtests to assess domains of attention, memory, visuospatial/construction and language ability.

1. The Preclinical Alzheimer Cognitive Composite [PACC, 23] which is a selection of 4 subtests weighted towards episodic memory. It comprises four components: the Logical Memory I and II [extracted from the Wechsler Memory Scale-Revised, 24], the Free and Cued Selective Reminding Test [FCSRT, 24, 25], the Wechsler Adult Intelligence Scale-IV (WAIS-IV) Coding subtest [24], and the Mini-mental State Examination [MMSE, 26].
2. The Binding in Neuropsychiatric Disorders Visual Short-Term Memory Binding Test [BIND-VSTMB, 27] is a web-based self-administered task that assesses conjunctive memory binding ability, the ability to integrate different pieces of information (e.g. a shape and its’ colour) into a cohesive memory representation [28].
3. The Four Mountains Task [4M, 29] is an allocentric spatial memory test. Memory for the topographical layout of four mountains within a computer-generated landscape (on iPad) is tested using a delayed match-to-sample paradigm.

The following measures will be employed to assess psychosocial parameters:

1. The Center for Epidemiological Studies-Depression Scale [CES-D, 30] will be used to assess depressive symptoms. The CES-D is a 20-item measure that asks participants to rate how often over the past week they experienced symptoms associated with depression, such as restless sleep, poor appetite, and feeling lonely. The State-Trait Anxiety Inventory (STAI) form Y [31] will be used assess anxiety symptoms. State anxiety items include: “I am tense; I am worried” and “I feel calm; I feel secure.” Trait anxiety items include: “I worry too much over something that really doesn’t matter” and “I am content; I am a steady person.”
2. Lifetime trauma, early life stress, post-traumatic stress symptoms will be assessed using the Life Events Checklist for DSM-5 [LEC5, 32] is a self-report questionnaire created to identify potentially traumatic events experienced by individuals throughout their lives. It evaluates exposure to 16 specific events recognized to potentially lead to PTSD or significant distress. Additionally, it includes an extra item to gauge any other exceptionally stressful event not covered by the initial 16 items. In addition, adverse childhood events before the age of 18 will be assessed using six items from Yang, Quach [33]. These items comprise yes/no responses to the following questions: ‘Before age 16, would you say your family during that time was pretty well off financially, about average, or poor?’; ‘While you were growing up, before age 16, did financial difficulties ever cause you or your family to move to a different place?’; ‘Before you were 18-year old, did you have to do a year of school over again?; ‘Before you were 18-year old, were you ever in trouble with the police?’; ‘Before you were 18-year old, did either of your parents drink or use drugs so often that it caused problems in the family?’ and, ‘Before you were 18-year old, were you ever physically abused by either of your parents?’.
3. Alcohol Misuse will be assessed using the Alcohol Use Disorder Identification Test [AUDIT, 34] is a 10-item alcohol harm screening measure developed by the World Health Organisation and adopted by several healthcare settings as well as other veteran studies [35, 36].

The following measures will be employed to assess lifestyle variables:

1. Sleep will be assessed using the Pittsburgh Sleep Quality Index [PSQI, 37] for sleep quality and the 10-item Berlin Sleep Questionnaire [BSQ, 38] for daytime sleepiness. The PSQI is an 8-item scale comprising subscales of sleep latency, sleep duration, habitual sleep efficiency and subjective quality. Scores range from 0–3 and are summed to yield a total score raging 0–12; higher scores indicative of worse overall sleep quality. The BSQ is a 10-item questionnaire that asks questions about snoring, daytime fatigue and breathing during sleep. Scoring is divided into categories and is contingent on participant responding. Category 1 is positive with 2 or more positive responses to questions 2–6; Category 2 is positive with 2 or more positive responses to questions 7–9; Category 3 is positive with 1 or more positive responses and/or a BMI>30; Final Results: 2 or more positive categories indicates a high likelihood of sleep apnoea.
2. Physical Activity will be assess using the International Physical Activity Questionnaire Short Form [IPAQ-SF, 39]. This includes a question on the duration and frequency spent sitting, walking, partaking in moderate activities and vigorous activities.
3. Current diet will be assessed by asking participants the following question: Compared to a month ago, have you managed to keep a healthy diet, for example eating fresh fruits and vegetables (response options: “Yes, I always have a healthy diet”; “Overall, my diet has improved since a month ago”; “Overall, my diet has been worse since a month ago”; “No, I always have an unhealthy diet”).
4. Smoking Status & History will be assessed by asking participants the following questions: Do you currently smoke? (“No, have never smoked”; “No, just have tried once or twice”; “No, stopped smoking”; “Yes, only occasionally”; “Yes, on most or all days”); How old were you when you first started smoking? (Age; “Do not remember”); How old were you when you stopped smoking? (Age; “Do not remember”) What did you usually smoke? (Cigarettes; Cigars; Pipe; None of the above; other).
5. Subjective Cognitive Complaints will be assessed by asking participant to response yes/no to the following questions: Have you noticed changes to your memory ability/ thinking ability (e.g. concentration, decision-making)/orientation to time (e.g. you confuse morning and evening)/orientation to space (e.g. you get lost more often in familiar places)?

Biological assessments.

Participants will be asked to fast from approximately 20:00 the evening before their research visit scheduled between 07:30–09:30. Fasting is to be confirmed verbally with the participant and any deviations noted. Participants will receive breakfast following the biological capture but are not offered caffeinated beverages until completion of neurophysiology.

Subject to funding, time constraints and expertise available at the research site, analyses will be conducted in-house or via external partners (participants are informed of and consent to the possibility of external collaboration for biological data analysis). All biological samples receive separate HTA identification numbers, preserving confidentiality. The use of biologically based markers for AD detection (blood- and saliva-based biomarkers, in particular) is a rapidly evolving research landscape [40]. Therefore, the following methods, procedures and biomarker targets may change to incorporate new technologies, methodological advances and future literature.

1. Blood sample processing. A total of 40ml of blood will be drawn primarily from antecubital vein into one 10 ml red top serum tube and three separate 10ml purple top EDTA vacutainers by suitably trained personnel for analyses of blood-based biomarkers including endocrinological and immune markers (e.g. estradiol, progesterone, testosterone levels, Il-6, TNFa), and AD-relevant biomarkers (e.g. Aβ-40, Aβ-42, BDNF, GFAP, NFL, brain derived tau, total tau, pTau181/217). Samples will be centrifuged at 4°C (preferred) or room temperature at 1500–3000 x g for 5–15 minutes until plasma and cells or until serum and clots are separated. The samples are then aliquoted in 0.5ml volume per aliquot and then stored at -80°C until down-stream biomarker analyses are performed. These processes occur immediately or at least within 2 hours of collection.
2. Saliva. Saliva ascertainment will involve the participant, via simple passive drool method collecting a morning and evening saliva sample, using the patented SimplOFy™ Saliva Collection Kits [Catalog Number SIMPL-302]. Spools will be used for analysis of AD-related saliva biomarkers (e.g. lactoferrin, amyloid beta and neurofilament-light-chain), cortisol and other steroid hormones and inflammatory markers. Participants complete the self-administered morning capture using the device at the research visit. They are asked to repeat this procedure at-home on the day of their visit and return the sample to the research team via pre-paid postage and packaging service that meet regulatory compliance. Following administration or receipt, devices are stored at −80°C until analyses are performed. Returned samples are processed and stored identically to research-visit samples.
3. Hair. Hair samples will also be collected at the morning time point, to evaluate endocrine and metabolic biomarkers expressed chronically. For this purpose, participants provide a strand of their hair (about 1/2 pencil thick) cut with scissors as close as possible to the scalp. The hair sample will be wrapped in tin foil and stored for future analysis.

Neurophysiological assessment.

Neurophysiological testing will be performed by a trained administrator and involve the use of single- and paired-pulse transcranial magnetic stimulation, in conjunction with percutaneous electrical stimulation of peripheral nerves. In addition to ruling out the exclusion criteria for TMS, the criteria from Rossi, Antal [41] will be identified before assessments. All stimulations will be delivered with the participant resting, with any trials containing pre-stimulus muscle contraction disregarded.

1. Electrical nerve stimulation. The maximum muscle compound action potential (Mmax) will be quantified by delivering 0.2 ms stimuli to the median nerve (DS7AH stimulator; Digitimer, Welwyn Garden City, UK). Electrodes will be placed ~2 cm apart over the nerve with the cathode positioned distal to the anode and ~2 cm proximal to the wrist. Single stimuli will begin at an intensity of 10 mA, and increase in 5 mA increments until the evoked potential amplitude plateaus. The intensity at the plateau will be recorded and one further stimulation will be delivered at 120% of this value to ensure a supramaximal stimulus. The Mmax will be recorded and used to normalise subsequent stimulations. Thereafter, a visual contraction threshold will be recorded as the lowest intensity needed to elicit a contraction of the first dorsal interroseous (FDI) muscle (approximately 12–18 mA).
2. Transcranial magnetic stimulation. Single and paired pulse magnetic stimuli of 1 ms duration will be delivered over the contralateral motor cortex to the non-dominant hand (postero-anterior intracranial current flow) with a figure of eight coil powered by a BiStim unit with a Magstim 2002 stimulator (The Magstim Company, Whitland, UK). Optimal stimulation location will be determined as the area of stimulation eliciting the greatest motor evoked potential (MEP) in the first dorsal interroseous (FDI). Resting motor threshold (rMT) will be quanitfied as the lowest stimulation intensity (% maximal stimulator output) eliciting a discernable MEP in ≥3 out of 5 stimulations.
3. Surface electromyography. Evoked potentials will be recorded via a bipolar configuration of Ag-AGCl electrodes placed over the FDI, with a reference electrode placed on the wrist. EMG will be amplified (×1000, 1902, Cambridge Electronic Design, Cambridge, UK), sampled at 5000 Hz, band pass filtered (20–2000 Hz, Power1401, Cambridge Electronic Design, Cambridge, UK).

The above methodologies will be combined to assess the following neurophysiological variables:

1. Corticospinal tract excitability: will be assessed with single-pulse TMS. The average motor evoked potential amplitude from 20 stimuli delivered at 120% rMT will be quantified and expressed in both mV as well as %Mmax to facilitate between-subjects comparisons.
2. Short-interval intracortical inhibition (SICI): ɣ-aminobutyric acid (GABA)-ergic inhibition within the motor cortex will be assessed with paired-pulse TMS. First, a subthreshold (80% rMT) pulse will be delivered 2.5 ms prior to a test pulse [120% rMT, 42]. The amplitude of the conditioned MEPs will be expressed as a % of unconditioned MEPs. Twenty conditioned MEPs will be compared with 20 unconditioned MEPs.
3. Intracortical facilitation (ICF): Facilitatory glutamatergic neurotransmission within the motor cortex will be assessed with paired-pulse TMS. First, a subthreshold (80% rMT) pulse will be delivered 12 ms prior to a test pulse [120% rMT, 42]. The amplitude of the conditioned MEPs will be expressed as a % of unconditioned MEPs. Twenty conditioned MEPs will be compared with 20 unconditioned MEPs.
4. Short- and long-latency afferent inhibition (SAI, LAI): The capacity to integrate antidromic sensory impulses with orthodromic motor impulses will be assessed with a combination of electrical nerve stimulation and TMS. First, an electrical stimulus will be delivered at 120% of the intensity that produces a visual contraction of the FDI [43, 44]. Thereafter, a single TMS pulse will be delivered 20ms after the electrical stimulation at 120% rMT for SAI, whereas the interstimulus interval will be 200 ms for LAI [45]. The amplitude of the conditioned MEPs will be expressed as a % of unconditioned MEPs. Twenty conditioned MEPs will be compared with 20 unconditioned MEPs.

In total, participants will receive 100 transcranial magnetic stimuli throughout the neurophysiological testing, this number is considered low risk by recent guidelines [41].

Military-specific psychosocial measures.

This study will pilot the use of items from the Deployment Risk and Resilience Inventory-2 questionnaires [DRRQ-2, 46] in military participants only. With support from veteran steering members and feedback from pilot veteran participants, the language of these items has been adapted to suit the British military context and the questioning timeframe will be adapted to include overall military service, rather than specific deployments. The purpose of including these items in the current study is to explore themes related to military service that may serve as the basis for themes and research questions in future qualitative studies. We will use and adapt the following questionnaires: Deployment environment; Combat Experiences; Exposure to Nuclear, Biological, or Chemical Agents; Post-battle Experiences; Support from Family/Friends and Relationships During Deployment. We will solicit feedback from participants regarding our adaptions and the questionnaire themes which include bullying, sexual and physical harassment as well as environmental and psychological exposures.