Corneal confocal microscopy (CCM)
For each participant, we capture 6 images (3 images per eye) containing the maximum number of nerves at the level of the sub-basal nerve plexus of the central cornea using a Heidelberg HRT III confocal laser-scanning microscope. This particular CCM has been demonstrated to be superior to other available instruments for corneal nerve assessment because of the higher contrast maintained to the edge of the field of view in all directions. Four metrics are used to quantify nerve fibres: Nerve fibre length (NFL), Nerve fibre density (NFD), Nerve-branch density (NBD) and Nerve fibre tortuosity (NFT).

Spectral-domain optical coherence tomography (Spectralis OCT)
Spectralis OCT is undertaken to quantify peri-papillary retinal nerve fiber layer (RNFL) thickness using the Spectralis 3.5-mm circle scan protocol with ART-Modus and eye tracking. Thinning of the inferior quadrant in RNFL is associated with increasing neuropathic severity (P = 0.03) and can distinguish between patients at high compared to low risk of foot ulceration (P = 0.0005)

Nerve conduction studies (NCS)
We perform NCS using a 2 channel EMG and NCS system (Dantec Dynamics Ltd, Bristol, UK).The assessments are conducted using surface stimulation and recording electrodes at skin temperature above 31oC. We record nerve conduction amplitude, velocity and latency of peroneal and tibial motor, and sural sensory nerves.

Autonomic Nerve Function studies (ANFS)
Cardiac autonomic function is measured using the ANX 3.0 autonomic nervous system monitoring device (ANSAR Medical Technologies Inc. Philadelphia, US) by performing spectral analysis of heart rate variability (HRV) during standard tests of cardiovascular reflexes. HRV during expiration and inspiration (E/I), Valsalva maneuver (VM), response to standing (30:15 ratio) and postural drop in systolic blood pressure in addition to an assessment of the low frequency area (LFa) to respiratory frequency area (RFa) ratio; an estimate of sympathovagal balance, will be established.

Quantitative sensory testing (QST)

Assessment of Vibration Perception Threshold (VPT) is measured using a Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham, UK). Cold and warm threshold testings are undertaken on the dorsum of the left foot using the MEDOC Q-Sense.