Exercise Intolerance in Post-COVID Patients

Absolute immune count will be assessed via BD TruCount in collected blood samples (before, immediately-, and 48h-post) in response to different exercise trials (high intensity interval training, moderate intensity continuous exercise and strength exercise) across the post-COVID subjects and age/sex matched controls. PBMCs will be analysed for distribution of B cells, NK cells, monocytes, CD4+ T cells, CD8+ T cells, with additional panels to capture plasma, effector and memory cells.

Metabolomic and cytokine profiling will be explored in collected blood samples (before, immediately-, and 48h-post) in response to different exercise trials (high intensity interval training, moderate intensity continuous exercise and strength exercise) across the post-COVID subjects and age/sex matched controls. Olink PEA platform and GC-MS at the Swedish Metabolomics Centre in Umea will be used and targeted analysis will include IL-6, TNF-a and IFN-g profiling.

Routine echocardiography wiil be performed to assess normal/abnormal heart structure and function in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up. A blinded observer will then quantify cardiac chamber dimensions and volumes, as well as the assessment of left ventricular systolic and diastolic function.

Routine spirometry wiil be performed to assess normal/abnormal lung structure and function in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up. The spirometry variables will be the forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), FEV% (FEV1/ VC MAX), maximum vital capacity (VC MAX), total lung capacity (TLC), and diffusing capacity of the lungs for carbon monoxide single breath corrected for Hb (DLCOc SB).

Non-invasive measurement of arterial stiffness via arteriography (Tensiomed) will be performed in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up. Higher pulse wave velocity (PWV) reflects higher degree of arterial stifness.

Head-up TILT test (HUTT) will be performed according to clinical guidelines to assess normal or abnormal autonomic nervous system regulation in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up. Specifically, hemodynamic measurements (systolic blood pressure, diastolic blood pressure and heart rate) will be measured non-invasively using NOVA software at supine position and at standing. Medical doctor will then classify the patients as follows:

1. Normal orthostatic reaction
2. Post-orthostatic tachycardia syndrome (POTS): development of symptoms with a sustained heart rate increase of ≥ 30 bpm or ≥ 120 bpm within 10 minutes of standing, with no significant blood pressure difference, i.e., SBP decreased by ≥ 20mmHg and DBP ≥ 10mmHg
3. Syncope: significant fall of HR or BP with loss of consciousness or inability to maintain posture
4. Orthostatic hypotension: any HR increase with SBP decrease of ≥ 20mmHG or DBP decrease of ≥ 10mmHG

The standard 6-min walk test will be performed to assess the physical function, including aerobic capacity in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up. Higher distance (in meters) covered by the participants reflects better physical function and aerobic capacity.

The gold standard Jama hydraulic hand dynamometer will be used to measure handgrip strength of the domiant hand to assess upper body muscular strength in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

The optimized carbon monoxide rebreathing method will be used to determine total hemoglobin, from which blood volume and plasma volume will later calculate using hemoglobin concentration [Hb] and hematocrit (Hct). This method will be conducted in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

Maximal isometric (120°/s) and isokinetic strength (60°/s and 210°/s) will be measured using Biodex isokinetic dynamometer to assess lower body muscular strength in the post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

Nerve conductions studies (NCS) will be conducted in the dominant lower and upper limb using the Cadwell Sierra Summit EMG system (Cadwell Industries, Inc., Kennewick, WA). Motor studies included the median, ulnar, fibular, and tibial nerves, and the compound muscle action potential (CMAP), distal latency (DL), conduction velocity (CV), and minimum F-wave latency will be recorded for all nerves. Sensory studies will include median, ulnar, radial, sural, and fibular nerves, and the sensory nerve action potential (SNAP) and CV will be recorded for all nerves. NCS will be performed in the post-COVID patients and age/sex matched controls at baseline and 1y follow-up

Needle electromyography (EMG) of 6 muscles (deltoid, biceps brachii, trapezius, anterior tibial, vastus medialis, and adductor magnus) will be performed using Sierra Summit EMG system in all participants at baseline and 1y follow-up. Multi-MUP analysis and routine EMG will be performed to record and assess MUPs qualitative and semi-quantitatively. Mean duration (simple MUPs), amplitude, and percentage of polyphasic MUPs will be recorded.

Sympathetic skin response (SSR) will be conducted to the dominant palm and sole using Sierra Summit EMG system to assess autonomic sympathetic function. The response will be recorded, and the latency determined from the onset of the stimulus artifact to first deflection from baseline. A response will be defined as absent if no reproducible deflection is recorded after consecutive and sporadic stimulations. This test will be performed in post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

Heart rate variability (HRV) using continuous electrocardiogram (ECG) will be recorded by a two-lead electrocardiograph using the Sierra Summit EMG system. The variation in R-R interval during normal breathing and during deep breathing will be recorded and the variability calculated as maximum heart rate minus minimum heart rate divided by average heart rate, expressed as a percentage. This test will be performed in post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

Tissue samples of the vastus lateralis muscle will be obtained under local anaesthesia using the percutaneous Bergström technique from the right leg. All muscle samples (~200 mg) will be immediately dissected free of any visible fat, excess blood and connective tissue, and then quickly frozen in liquid nitrogen. Samples were stored at -80°C until the analyses. Muscle biopsies will be collected in post-COVID subjects and age/sex matched controls at baseline and 1y follow-up.

Cardiopulmonary exercise testing (CPET) will be used to assess exercise capacity as the peak volume of oxygen consumption (VO2 peak) in all participants at several timepoints in this study. First at baseline, then 48h after each exercise trial and finally at the 1y follow-up.

Multifactional Fatigue Inventory (MFI) validated questionnarie will be administrated to assess fatigue exaggeration in response to 3 different exercise trials (high intensity interval training, moderate intensity continuous exercise and strength exercise) between the post-COVID subjects and age/sex matched controls. MFI is a 20-item self-report instrument designed to measure fatigue. It covers the following dimensions: General Fatigue, Physical Fatigue, Mental Fatigue, Reduced Motivation and Reduced Activity. Higher scores reflect higher fatigue levels.

2 groups x 3 exercise sessions x 3 timepoints per exercise (pre, post and 48h post)

Profile of Mood States (POMS) instrument will be administrated to assess different dimensions of mood states in response to 3 different exercise trials (high intensity interval training, moderate intensity continuous exercise and strength exercise) between the post-COVID subjects and age/sex matched controls. POMS measures six different dimensions of mood swings over a period of time. These include: Tension or Anxiety, Anger or Hostility, Vigor or Activity, Fatigue or Inertia, Depression or Dejection, Confusion or Bewilderment.

Higher scores reflect increased levels mood states (i.e. higher tension level).

2 groups x 3 exercise sessions x 3 timepoints per exercise (pre, post and 48h post)

The Somatic and Psychological HEalth REport (SPHERE) is a 34-item self-report questionnaire that assesses symptoms of mental distress and persistent fatigue. Scores from 12 selected questions will be used to calculate the subscale of SOMA reflect physical fatigue (6 questions) and PHYCH metnal distress (6 questions) respectively. SPHERE instrument will be administrated to assess mental stress and persisent fatigue in response to 3 different exercise trials (high intensity interval training, moderate intensity continuous exercise and strength exercise) between the post-COVID subjects and age/sex matched controls.

Higher SOMA and PHYCH scores reflect increased levels persistent physical fatigue and mental stress respectively.

2 groups x 3 exercise sessions x 2 timepoints per exercise (pre and 48h post)