Diaphragmatic and Sternocleidomastoid Muscle Recruitment at Various Inspiratory Loads With and Without Breathing Instruction in Healthy Adults
Conditions: Diaphragm
Sponsor: Hong Kong Metropolitan University

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Study protocol 
Diaphragmatic and sternocleidomastoid muscle recruitment at various 
inspiratory loads with and without breathing instruction in healthy adults 
 
Objectives 
(1) 
to investigate the relationship between sternocleidomastoid muscle recruitment and 
diaphragm thickness fraction during increasing inspiratory resistance in healthy adults. 
(2) 
to compare diaphragmatic and sternocleidomastoid muscle recruitment patterns with and 
without a standard diaphragmatic breathing instruction with increasing inspiratory 
resistance in healthy adults. 
 
Method 
Ethical committee approval 
Ethical committee approval was granted by the Research Ethics Committee of Hong Kong 
Metropolitan University (ethics approval number: HE-OT2023/13). 
Sample size calculation 
Sample size will be determined by repeated-measures analysis of variance (ANOVA) using 
G*Power 3.1. Considering an effect size of 0.3, an alpha error probability of 0.05, a power of 
0.80, the estimated sample size is 30 participants. An additional 20% of the cohort will be 
recruited to compensate for the predicted sample size attrition. The final estimated sample size 
will be at least 36 participants. 
Participants 
Participants 18 years of age with normal health will be recruited and screened for any of the 
following ‘exclusion’ criteria: (1) participants with known cardiovascular, pulmonary, 
musculoskeletal or psychiatric disorders; (2) pregnancy; (3) with respiratory symptoms (from 
flu or other respiratory infection during the 2 weeks prior to measurements. 
Procedure  
Upon arrival at the laboratory, eligible participants will first perform a standard spirometry lung 
function test. The maximum inspiratory pressure (MIP) will be determined with a pressure 

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threshold inspiratory loading device (POWERbreathe, K2). Each participant will then be 
instructed to use the device to perform, in random order, 10 breaths at an inspiratory intensity 
of 30%, 40%, 50%, 60%, 70%, and 80% MIP.  A rest period of 15 minutes will be allowed 
between each inspiratory pressure set of 10 breaths. Simultaneous surface electromyographic 
(sEMG) recording of the right sternocleidomastoid muscle and ultrasound of right 
diaphragmatic thickness will be conducted during each breath. 
The entire recording procedure will be repeated on a separate day. During the repeat 
measurements, participants will be provided with specific breathing instruction to focus on the 
use of the diaphragm and to inhale air to the lower part of the chest. Practice trials will be 
conducted to ensure participant’s understanding of diaphragmatic breathing. 
 
Measurements 
Spirometry respiratory function  
Forced vital capacity, forced expiratory volume in 1 second, and MIP will be measured 
following the American Thoracic Society guideline protocol. 
Diaphragmatic contraction  
During each of the 60 breaths, thickness of the right diaphragm at the end of each tidal 
expiration and at the end of each forced inspiration will be measured using diaphragmatic 
ultrasonography (Mindray M9, Shenzhen, China). The recorded thickness over each set of 10 
breaths will be averaged. The strength of diaphragmatic contraction will be reflected by the 
diaphragmatic thickening fraction (DTf), which is calculated as a ratio of [the difference 
between mean diaphragmatic thickness at the end of each inspiratory pressure and mean 
diaphragmatic thickness at the end of tidal expiration / the diaphragmatic thickness at the end 
of tidal expiration] x100%.  
Recruitment of the sternocleidomastoid muscle 
A Noraxon Ultium wireless sEMG system (Noraxon USA, Inc., Scottsdale, AZ, USA) will be 
used to detect the muscle recruitment activity of the right sternocleidomastoid muscle. On the 
2 days of data collection, sEMG data over each set of 10 breaths will be averaged for data 
analysis. 
Self-perceived exertion level 

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Self-perceived exertion level at the end of each 10-breath set at each inspiratory pressure, will 
be also recorded using the Borg’s scale of Rate of Perceived Exertion. 
 
Statistical analysis  
All data will be analyzed using the IBM SPSS Statistics for Windows, Version 25.0 (Armonk, 
NY: IBM Corp).  
Changes in DTf, muscle activity of sternocleidomastoid and fatigue during each inspiratory 
protocol will be analyzed using repeated-measures ANOVA.