The Effect of Aerobic Exercise on Immune Biomarkers and Symptoms Severity and Progression in Patients With COVID-19: A Pilot Randomized Control Trial

Evaluative procedures There were two main dependent variables including blood immune markers and severity of respiratory symptoms. These measurements were collected at the baseline after one week and 48 h after the end of the exercise program (two weeks).

A lab technician was asked to visit the patient at home (quarantine). The lab technician wore special protective equipment recommended by WHO. Three visits were performed, one at the beginning of the research procedures, one week after, and the last visit at the 48 hours after the end of the exercise program (two weeks). The technician collected blood and saliva samples to be analyzed.

Blood sample collection Blood samples were taken in the morning (8:30-9:30). 10mL of venous blood was collected. Participants were asked to stop any exercise for at least 24 hours before blood sampling. Also, participants were asked to stop eating any food or liquid from 22:00 the prior day of measurement. Samples were collected in vacutainer tubes with sodium ethylenediaminetetraacetic acid (EDTA) for plasma separation. The blood was centrifuged at 3,000 rpm for 15 min at 4◦C. We measured total lymphocytes, leukocytes, and monocytes from total-blood samples utilizing a multichannel hemocyte analysis system (SE-9000; Sysmex Corp, Hyogo, Japan). The concentrations of IL-6, IL-10, and TNF-α were analyzed by using ELISA commercial kits assay (R&D Systems, Minneapolis, USA) following the manufacturer's instructions for analysis on an EZ-Reader microplate reader at 450 nm. The samples were stored at -20◦C for further analysis.

Saliva sample collection A saliva sample was collected to measure the salivary IgA-S concentration. The saliva sample was taken without any saliva stimulation methods, the participant was asked to rinse their mouths with distilled water and to evacuate their mouth just before collection. We used the passive drainage method for the collection, in which the participant slightly flexed their head forward to allow the saliva to move into a sterilized and pre-weighed Falcon tube for 5min. The weight of tubes were measured again following collection, to estimate the volume and the saliva flow rate. The tubes were weighed with 0.1mg accuracy with proposed saliva density as 1.0 g.mL-1. The samples were stored at -80◦C for further analysis. The S-IgA concentration were analyzed utilizing commercial ELISA kits (IgA Salivary, DRG, Minneapolis, USA). The IgA-S secretion rate (ng/min) was measured by multiplying the whole concentration of IgA-S present in the mucosal surface per unit of time by the saliva flow rate (mL/min).

Wisconsin Upper Respiratory Symptom Survey The Wisconsin Upper Respiratory Symptom Survey (WURSS) is an empirically derived patient-oriented illness-specific quality-of-life evaluative outcomes instrument. The development process of this survey was described in detail by Barrett et al. WURSS-24 is designed to evaluate the negative effect of acute upper respiratory infection, presumed viral (the common cold). Its a valid and reliable measurement tool to evaluate the measure items and domains that change over time including influenza-like illness symptoms of headache, body aches, and fever. The participants were asked to fill the survey before starting the study and the 2 times/week.