Table 2 Outcome measures

Primary outcome:

The change in ATP content in skeletal muscle biopsy specimens

Secondary outcomes:

1. Health-related quality of life

- The Quality of Life in Neurological Disorders (NeuroQol), evaluates and monitors the physical, mental and social effects experienced by adults and children living with neurological conditions [13]

- Newcastle Mitochondrial Quality of life questionnaires (NMQ), psychometric evaluation of mitochondrial disease-specific health-related quality of life [14]

- Visual Analogue Scale (VAS) for pain and most bothersome symptom, measuring intensity of symptoms [15]

2. Reported perceived fatigue

- Fatigue Impact Scale (FIS) assesses symptoms of fatigue. Evaluates the effect of fatigue on daily life for cognitive, physical and psychosocial functioning [16, 17]

- Fatigue Severity Scale (FSS) will be used to measure the impact and severity of fatigue [18]

3. Symptom-limited cardiopulmonary fitness (optional)

Performed on a recumbent cycle ergometer with resistance consistently increased via a ramp protocol at 5 to 15 watts/minute. Variables measured at peak (volitional exhaustion) included:

- VO₂, VCO₂, anaerobic threshold (AT), pulmonary ventilation (VE), respiratory exchange ratio (RER) and breathing frequency (F)

- Work rate (power, measured in watts)

- Heart rate (HR), stroke volume (SV), cardiac output (Q), and arteriovenous oxygen difference (a-VO₂ diff)

- Rate of perceived exertion [19]

- Blood lactate (mmol/L)

- Blood oxygen saturation (SpO₂) (estimated via pulse oximetry)

4. Disease burden

- Newcastle Mitochondrial Disease Adult Scale (NMDAS), a semi-quantitative clinical rating scale to assess disease burden for all forms of mitochondrial disease [20]

5. Upper and lower limb function, balance and walking

- 6-Minute Walk Test (6MWT) assesses aerobic capacity and endurance, measuring distance covered over 6 min comparing changes in performance [21]

- 10-Metre Walk Test (10MWT), assesses walking speed in metres per second over a short distance and can help determine functional mobility, gait and vestibular function [22]

- Mini Balance Evaluation Systems Test (Mini-BESTest), to detect balance impairments [23]

- 9 Hole Peg Test (9HPT), to measure finger dexterity [24]

- 30-second Sit To Stand (STS) to evaluate lower extremity functional strength [25]

- Scale for the Assessment and Rating of Ataxia (SARA) assesses different impairments in cerebellar ataxia [26, 27]

6. Skeletal muscle analyses

- ATP/ADP ratio via luminescence assay

- mtDNA copy number as a marker of mitochondrial density using q-RT PCR.

- Respiratory chain deficiency using sequential cytochrome c oxidase and succinate dehydrogenase (COX/SDH) histochemistry and quadruple immunofluorescence

- mtDNA heteroplasmy via q-RT PCR or pyro sequencing

Exploratory outcomes

- Investigation of mitochondrial disease markers via Enzyme-Linked Immunosorbent Assay (ELISA) (Fibroblast Growth Factor 21) (FGF-21) and Growth Differentiation Factor 15 (GDF15)

- NAD⁺/NADH ratio via luminescence assay

- mtDNA heteroplasmy in blood and urine via q-RT PCR or pyro sequencing

- Assessment of diabetes markers (insulin, HbA1C, C-peptide and glucose)

- Instrumented gait analysis, to assess differences in gait parameters [28]

- Assessment of habitual physical activity and sleep using wrist-worn accelerometers worn continuously over 7 consecutive days (optional)

- Assessment of participant self-reported symptoms captured via daily Visual Analogue Scales (VAS) [24] for pain and most bothersome symptom and fatigue via Daily-Fatigue Impact Scale (D-FIS) [29]

- Skeletal muscle mitochondrial morphology via Electron Microscopy (EM) will be performed for a subset of participants where sufficient tissue is available

- Quantitative proteomics analysis, including assessment of PGC-1α and SIRT-1 mediated mitochondrial biogenesis, carried out on a sub-set of samples, should funding and tissue be available.