Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Endurance exercise test
• Definitions and calculations
• Data

Workflow and sampling

Step	Procedure performed	Equipment	Data collected
1	Familiarization with equipment	treadmill	-
2	Body weight measured prior to each test	scale	body weight
3	Endurance exercise test	treadmill	duration, distance, work

Equipment and supplies

• scales
• six-lane treadmill (Columbus Instruments, Columbus OH)

Reagents and solutions

• cleaning solution

Acclimation to test conditions

Mice were allowed one week to become accustomed to the housing facility before starting the exercise test.

Procedure: Endurance exercise test

Mice perform two graded exercise endurance tests on a 6-lane treadmill, separated by 48 h. For each mouse, the test average is taken.

1. Mice are first familiarized for 2 days: day 1 running for 10 min, 10° incline, at a speed of 9 m/min; day 2 running for 10 min, 10° incline, at a speed of 11 m/min.
2. Mice are tested on days 3 and 5 after weighing. The exercise capacity protocol is as follows: 9 min, 0° incline, speed 9 m/min --> 9 min, 5° incline, speed 10 m/min --> thereafter speed was increased 2.5 m/min every 3 min, grade was increased 5° every 9 min to a maximum grade of 15°.
3. Testing is terminated when mice maintain contact with the grid for more than 15 s continuously or can no longer be motivated to run.

Definitions and calculations

• work: calculated as a product of body weight and vertical distance, where vertical distance = distance run x sin(theta), where theta is equal to the angle of the treadmill

Data collected by investigator

• body weight
• duration
• distance
• work