Evidence suggests that gut microbiota contributes to the development of atherosclerosis and therefore CVDs. Exercise has been extensively reported to be protective against CVDs due to modification of body fatness, metabolic risk factors and cardio-respiratory fitness. It remains unclear whether impact of exercise on CVD risk can be at least in part explained by changes in the microbiota. It also remains to be investigated whether modification of appetite and appetite hormones due to participation in exercise are related to changes in the gut microbiota composition. Therefore, this study will be conducted in overweight, healthy, sedentary women and will investigate the effect of a 4-week moderate intensity exercise programme on gut microbiota composition and metabolic activity, plasma concentrations of fasting and postprandial lipids, glucose, insulin and inflammatory markers, fasting and postprandial gut appetite hormones, subjective appetite measures and cardio-respiratory fitness.

Study Design: This is a randomized controlled study with a parallel design. Healthy, sedentary females aged between 18-45 years old will be recruited by means of an advertisement leaflet and word of mouth. Eligible participants will be enrolled in the study and will be randomly allocated to either exercise intervention (EX) or control (C) groups. The EX group will participate in a 4-week exercise intervention and the C group will be asked to continue with habitual life style for 4 weeks. Before and after 4 weeks of the intervention, participants from both groups will be asked to conduct a submaximal exercise test and a 7-hour experimental trial during which blood samples will be collected and metabolic rate will measured before and after consumption of standardized breakfast and lunch. Prior to the first 7-hour trial, participants will be asked to record diet for 3 days and replicate this diet prior to the second 7-hour trial. During these 3 days participants will provide fresh stool samples. Participants allocated to control group will have the option to participate in exercise intervention. A subset of the participants will repeat a 7-hour experimental trial and provide fresh stool and urine sample after 7 days of detraining.

Methods:

Preliminary Health Check: Participants will undertake a health screening questionnaire on the day of recruitment. Participants will also need to fill in Physical Activity Readiness Questionnaire (PARQ). Only participants who answered "no" to all questions in PARQ will conduct the submaximal exercise test.

Anthropometry: All participants will have height, weight and waist circumference measured at baseline and 4 weeks after the intervention.

7-hour Experimental Trial: Participants will attend the metabolic suite (New Lister Building, Glasgow Royal infirmary) in a fasted state at approximately 9.00 am. Participants will be instructed to lie supine on the couch, with the head resting on the pillow provided. Resting metabolic rate will be measured for 20 minutes and fasting blood sample will be obtained. A breakfast meal will be consumed immediately after the fasting sample and a lunch meal after 4 hours. Blood samples will be taken throughout a cannula hourly after breakfast and lunch consumption. Following each blood sample, metabolic rate will be measured for the duration of 20 minutes. Prior each blood sample, participants will fill in appetite questionnaires. Blood samples will be analysed for plasma lipids, glucose, insulin, inflammatory markers and gut appetite hormones using standard existing procedures.

Feacal sample collection & analysis: A disposable stool sample collection kit will be provided prior to the 7-hour trial day. Once collected, the container is sealed and placed in a larger sealable bag with a reducing sachet to remove oxygen and maintain anaerobic bacteria. Stool samples will be measured for changes in gut microbiota based on 16S rRNA amplicon sequencing, bacterial functional capacity and targeted bacterial metabolites (short chain fatty acids, sulphide, ammonia).

Exercise Training Programme: Exercise training will consist of 4 weekly sessions of endurance type exercise (cycling and running) lasting for 30, 40, 50, 60 minutes in the week 1, 2, 3 and 4 respectively. The time and day of each training trial will be agreed between the investigator and the participant and will be based around the participant's availability. All exercise sessions will be supervised by a researcher.

Submaximal Exercise Test: Participants will exercise on treadmill at gradually increasing intensities until 85% of maximal heart rate. During the last minute of each of the intensities heart rate will be measured and expired air will be collected by Douglas bag technique and analysed for oxygen consumption and carbon dioxide production. Relation between heart rate and rate of oxygen consumption will be used to predict maximal oxygen consumption, a marker of cardio-respiratory fitness.

Inclusion Criteria:

• Healthy, sedentary females, BMI>25 kg/m2, on a stable body weight for the last 3 months

Exclusion Criteria:

• Acute or chronic illness, use of antibiotics the past 3 months, smoking