Layout 1 Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 The diaphragm is the main respiratory muscle, working in coordination with other accessory muscles (such as intercostals), to support the work of breathing. Beyond its respiratory function, research highlights the diaphragm’s crucial role in trunk stability and posture.1 Located at the superior border of the “abdominal canister” the diaphragm works in synergy with the pelvic floor and abdominal muscles to modify or maintain intra-abdominal pressure during static and dynamic tasks, thus increasing spinal stability.1-5 Its unique anatomy with its crurae attached to the lumbar vertebrae is also considered to contribute to spinal stability.6,7 Although Rehabilitative Ultrasound Imaging (RUSI) was initially proposed for the assessment of the abdominals, pelvic floor and multifidus muscles,8,9 over the past dec- ade, RUSI has gained significant popularity among respi- ratory therapists, Physical and Rehabilitation Medicine (PRM) professionals, physiotherapists, and researchers, offering a safe and cost-effective method to assess dia- phragm function as opposed to traditional, high-cost, in- vasive or other non-invasive assessment methods (i.e. dynamic magnetic resonance imaging, fluoroscopy, elec- tromyography, trans-diaphragmatic pressure assessment etc.).10,11 Utilizing brightness- or motion-modulation, dia- phragmatic RUSI is applied to assess diaphragm thickness and excursion parameters in critical care,12 cardiac,13,14 and Abstract Beyond respiration, the diaphragm contributes to trunk stability and posture. Rehabilitative Ultrasound Imaging (RUSI) assesses Diaphragm Thickness (DT) and Excursion (DE), but evidence on reliability across novice examiners is limited. This study investigated intra- and inter- tester reliability of diaphragmatic RUSI across novice examiners in healthy women and compared DT and DE between parous and nulliparous participants. Healthy parous and nulliparous women participated. Exclusion criteria included pregnancy, chronic conditions, and BMI > 30 kg/m2. Two physiotherapists, novices in RUSI, measured DT during full inspiration and expiration and DE during quiet breathing. An independent examiner calculated the Thickening Fraction (TF). Three within-session measurements were taken for intra-tester reliability, whereas, for inter-tester reliability, agreement between mean measurements was analyzed via Intraclass Correlation Coefficients (ICC) and 95% Confidence Intervals (CIs). Independent samples t-tests assessed DT and DE differences between groups. Twenty women (33±14.3 years, BMI: 23.4±3.3) participated. Intra-tester reliability was moderate to good for DT (ICC:0.511-0.691) and very good for DE (ICC:0.700-0.756). Inter-tester reliability was very good for DE (ICC=0.748), and moderate for DT (ICC: 0.573-0.706). TF reliability was poor. Only one tester found higher inspiratory DT in parous women (p<0.01). Novice examiners can reliably measure DT and DE using RUSI, supporting its use in rehabilitation. Further standardization is required to enhance RUSI practices and additional research to clarify parity-related diaphragm changes. Key Words: diaphragm, ultrasound imaging, ultrasonography, RUSI, reliability study. Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 Intra- and inter-tester reliability of diaphragm thickness and excursion measurements of healthy women using rehabilitative ultrasound imaging across novice examiners Anastasia Skoura,1 Evdokia Billis,1 Maria Andriopoulou,2 Elena Drakonaki,3 Dimitra Tania Papanikolaou,1 Maria Tsekoura,1 Eleni Kortianou,4 Ioannis Maroulis5 1Laboratory of Clinical Physiotherapy and Research, Department of Physiotherapy, School of Health Rehabilitation Sciences, University of Patras, Greece; 2General Hospital Asklipieio Voulas, Greece; 3Anatomy Department, Medical School, University of Crete, Heraklion, Greece; 4Department of Physiotherapy, University of Thessaly, Greece; 5Department of Medicine, University of Patras, Greece. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 107 - Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 pulmonary rehabilitation,15,16 as well as within the muscu- loskeletal field.17,18 Diaphragm thickness refers to the shortening and thickening of the diaphragm as the muscle fibers contract and relax during breathing, while excursion describes its craniocaudal displacement along its axis dur- ing the respiratory cycle. Indeed, reduced diaphragm thickness and lower diaphragm mobility have been ob- served amongst individuals with non-specific low back,19- 21 and lumbopelvic pain,22 compared to healthy controls. In postpartum populations, Kharaji et al.23 also found re- duced diaphragm excursion in women with lumbopelvic pain compared to asymptomatics, during a low postural demanding task. It is, therefore, not unreasonable to as- sume that diaphragm thickness and excursion, measured via Ultrasound (US) may have a central role within respi- ratory and musculoskeletal rehabilitation. Previous studies confirm strong intra-tester and inter-tester reliability of diaphragm thickness and excursion measure- ments among clinicians with varying backgrounds and ex- perience levels with ultrasonography.24-33 However, a crucial concern involves the ability to consistently meas- ure diaphragm thickness and excursion, especially among novice (in RUSI) physiotherapists. Given the rapidly in- creasing interest of RUSI across physiotherapy, this study aims to fill this knowledge gap, by assessing the intra- tester and inter-tester reliability of physiotherapists’ US measurements of diaphragm thickness and excursion ac- ross a healthy women sample. A secondary objective is to explore any differences in diaphragm measurements among healthy nulliparous and parous women, as this con- cept remains under-researched in relation to postpartum recovery and rehabilitation. Materials and Methods Study design and registration This reliability study was carried out from May to October 2023 following the Guidelines for Reporting Reliability and Agreement Studies (GRRAS).34 The Helsinki Decla- ration and human experimentation ethical requirements were respected.35 The study was approved by the Ethics Committee of the University of Patras (number 15543/12- 04-2023). All participants signed an informed consent form before participating in the study. Sample size calculation An a priori sample size calculation was performed using GPowerSoftware 3.1.9.7. A correlation coefficient of 0.6 was chosen, representing a strong association between measurements based on previously reported values.27 With a significance level of 0.05, a power of 0.80, and a one- tailed hypothesis, the sample size calculation determined a minimum of 15 subjects necessary to detect the specified correlation of 0.6 between intra-tester and inter-tester measurements. Subjects A convenience adult women sample around the broader university campus area was invited to participate in the study through advertisements and university announce- ments. Inclusion criteria were healthy women, nulliparous and parous, aged 18-60. Exclusion criteria were previous diagnosis of severe chronic respiratory conditions (i.e., COPD, chronic bronchitis etc.), Body Mass Index (BMI) > 30 kg/m2 that could challenge US imaging, previous major abdominal surgery, pregnancy, and neuromuscular dis- orders that could alter diaphragm behavior and function. Testers and training procedure Two physiotherapists, both experienced clinicians (over 7 years of clinical musculoskeletal and respiratory experi- ence), but novices in diaphragmatic RUSI, participated in a two-day training session led by a consultant radiologist, specializing in musculoskeletal sonographic imaging. Training was followed by several hours of independent and joint practice between the radiologist and physiother- apists. Throughout the study assessments, the physiother- apists had access to the radiologist for consultation whenever they were uncertain about a measurement. Diaphragm measurement procedure All subjects received US assessment using the Versana Active™ 2-D ultrasound system (GE HealthCare, Chi- cago, Illinois, US) in the semi-recumbent position (trunk elevated by 35°), hands positioned behind the head, and knees bent (60º of flexion). Diaphragm Thickness (DT) was assessed by the examiners at the zone of apposition of the right hemi-diaphragm, using a high-resolution linear transducer at 10 MHz in B-mode. The scan was performed over a marked area (Figure 1A), as described in Boussuges et al.36. The subject was asked to fully exhale and then inhale as deeply as possible, aiming at full chest and abdominal expansion. DT was measured in frozen B-mode in dual split screen (capturing two im- ages), at the end of full inspiration and full expiration (Fig- ure 1B). Three consecutive measurements were taken during each session. After the assessment, an independent researcher calculated and documented the thickening frac- tion according to the following formula: Thickening Fraction (TF) = (Thickness at End Inspiration - Thickness at End Expiration) / Thickness at End Expiration TF refers to the percentage of change in diaphragm thick- ness between inspiration and expiration, indicating the ex- tent of diaphragmatic contraction and relaxation during the respiratory cycle. TF can either be expressed as a per- centage (multiplied by 100%) or as a quotient from the given fraction, which was employed in this case. Diaphragm Excursion (DE) was assessed at the right hemi- diaphragm, through the liver acoustic window, using a high- resolution curvilinear transducer at 4 MHz in M-mode. To locate the best position for optimal visualization of the pos- terior hemidiaphragm displacement, B-mode was utilized to scan over the marked skin area (Figure 2A), as described in Boussuges et al.36 Once visualization of the right hemi- - 108 - Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 diaphragm with B-mode was achieved, M-mode was uti- lized to display the craniocaudal displacement of the dia- phragm through the respiratory cycle, appearing as a hyperechoic line. The subject was asked to inhale and ex- hale naturally, allowing free movement of the chest and ab- domen and the image was captured and measured after 1-3 respiratory cycles (Figure 2B). Three consecutive measure- ments were taken during each session. Reliability testing For intra-tester reliability, anatomical guide points were marked with a skin marker independently by each tester according to literature recommendations.36 These mark- ings were erased after each tester completed their meas- urements. Each tester performed six consecutive measurements, three for DT and three for DE with a two- minute resting interval between each measurement. Tester 1 initiated the measurements for half of the sample, while Tester 2 measured the other half first. For inter-tester reliability, the other physiotherapist re- peated the measurements during the same session; three DT and three DE measurements as previously described were measured again in random order. Examiners and sub- jects were blinded to all measurements taken, with post-it - 109 - Figure 1. Diaphragm thickness assessment. A) Position of the probe over the marked area, delineated by anatomical guide points. These points indicate the region 0.5 to 2.0 cm beneath the phrenico-costal sinus, positioned between the 8th and 9th intercostal spaces and extending between the anterior and mid-axillary line. Probe position is perpendicular to the chest wall. B) Caliper placement. The diaphragm is identified as a hypoechoic area enclosed between two parallel echogenic lines, the diaphragmatic pleura and the peritoneal fascia. Calipers are placed on the two hyperechoic lines (diaphragmatic pleura and peritoneal fascia) and the distance is calculated automatically by the machine’s built-in digi- tal measurement system. Two measurements were taken per respiratory cycle (end of full expiration and full inspiration) using a dual split-screen in B-mode. The image displays diaphragm thickness at full inspiration (left) and full expiration (right). Measurements were repeated across three consecutive cycles, with a two-minute interval between efforts. Figure 2. Diaphragm excursion assessment. A) Position of the probe over the marked area, delineated by anatomical guide points. These points indicate the low intercostal area/ area below the subcostal margin, between the mid-clavicu- lar and the mid-axillary line. The probe is pointed medially, dorsally and in a cephalad direction. B) Excursion meas- urement in M-mode. Calipers are placed at the foot of the slope and at the apex of the hyperechoic diaphragmatic line and their distance is calculated automatically by the machine’s built-in digital measurement system. Each image was captured during quiet breathing, typically after 1 to 3 full respiratory cycles, depending on the subject’s respiratory rate and how many cycles fit within the image frame. Measurements were taken based on the clearest excursion trace. Image B shows one complete respiratory cycle and a second cycle that ends at inspiration, representing an incomplete trace. Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 stickers placed over the tables displaying distance values at the upper and lower left edges of the US screen. Fol- lowing examination, an independent researcher blinded to the subject and examination procedure extracted and doc- umented all variables for each subject in a spreadsheet software. The mean of 3 repeated measurements for each testing variable was calculated. Statistical analysis Descriptive statistics, including measures of central ten- dency (range, mean, median, and standard deviation values) as well as frequencies and percentages, were em- ployed for interval and categorical variables, respectively. A Kolmogorov–Smirnov test was used to assess normal- ity. Differences between testers were reported utilizing paired samples t-tests, while differences across parous and nulliparous women were reported utilizing independent samples t-tests. Intra-tester and inter-tester reliability were estimated utilizing an Intraclass Correlation Coefficient (ICC) and 95% Confidence Intervals (CIs) according to literature recommendations.37 Intra-tester reliability was assessed with a two-way mixed effects model (ICC3,1) and inter-tester reliability with a two-way random effects model (ICC2,1). The interpretation of ICC values followed established guidelines;38 thus, values <0.40 were classified as poor, those between 0.40 and 0.59 as weak, between 0.60 to 0.74 as good, and values ranging from 0.75 to 1.00 were considered excellent. The standard error of measure- ment (SEM), indicating average amount of error of in- dividual measurements, was calculated using the formula . The smallest detectable dif- ference (SDD), representing the threshold beyond which a real change in measurement is considered, was cal- culated using the formula for a 95% CI. Statistical analysis was carried out utilizing SPSS (version 28.0). Results Twenty women aged 19-59 participated in the study; 11 nulliparous (55%) and 9 parous (45%). The sample’s so- ciodemographic characteristics are presented in Table 1. Statistically significant differences in age across groups were found, as parous women were older than nulliparous women. Each tester captured and measured 180 images. DT, DE and TF descriptives are presented in Table 2 - 110 - Table 1. Sociodemographic characteristics of the sample. Women sample (n=20) Nulliparous (n=11) Parous (n=9) Group differences Mean (SD) P-value Age (years) 33.1 (14.29) 24.3 (11.32) 43.8 (9.4) <0.001* Height (cm) 165.0 (6.6) 165.1 (5.7) 164.8 (8.0) 0.92 Weight (kg) 63.7 (9.8) 63.8 (10.1) 63.5 (10.0) 0.95 BMI 23.4 (3.3) 23.5 (4.1) 23.3 (2.2) 0.88 SD, standard deviations. * Statistically significant differences between groups. Table 2. Diaphragm parameter measurements across the two testers. Parameters Tester 1 Tester 2 P-value Mean (SD) Inspiratory Thickness (cm) 0.38 (0. 06) 0.40 (0.09) 0.224 Expiratory Thickness (cm) 0.24 (0.05) 0.23 (0.07) 0.706 Thickening Fraction 0.62 (0.31) 0.78 (0.42) 0.106 Excursion (cm) 4.66 (1.23) 4.28 (1.16) 0.058 SD, standard deviations. Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 whereas, Table 3 summarizes descriptive statistics across nulliparous and parous women. No statistically significant differences were found in DT, TF, or DE between testers across the sample (p>0.05). No statistically significant dif- ferences in diaphragm parameters were found across nul- liparous and parous women except for Tester 2’s inspiratory thickness measurements, where a statistically significant difference was evident (p<0.01). Intra-tester reliability Intra-tester reliability results for each tester are presented in Table 4. Tester 1 demonstrated a moderate to good agreement for DT measurements, both in inspiratory (ICC=0.511) and expiratory phases (ICC= 0.617). Tester 2 exhibited good intra-tester agreement, with inspiratory and expiratory DT yielding slightly higher agreement co- efficients (ICC=0.685, and ICC=0.691, respectively), compared to Tester 1. SEM calculations revealed rel- atively low variability in inspiratory and expiratory thick- ness measurements for both, Tester 1 (with SEM values of 0.045 and 0.032, respectively) and Tester 2 (with SEM values of 0.055 and 0.045, respectively). SDD, represent- ing the threshold beyond which a genuine change in meas- urement is considered, was acceptable, falling within the expected range of measurements (between 0.12cm and 0.15cm for inspiratory, and 0.09cm and 0.12cm for expi- ratory thickness). For DE, both testers demonstrated very good intra-tester agreements (ICCs>0.700). For Tester 2, US DE measure- ments demonstrated low variability, with small SEM and SDD (0.045cm and 0.12cm respectively). Tester 1 exhib- ited a moderate level of precision, with slightly larger SEM and SDD values (0.66cm and 1.83cm, respectively). TF revealed relatively low intra-tester agreement for both Tester 1 (ICC=0.481) and Tester 2 (ICC=0.501), larger SEM (0.276cm and 0.367cm, respectively), and SDD values (0.764 cm and 1.018 cm, respectively). Inter-tester reliability Inter-tester reliability results across the two testers are pre- sented in Table 5. Inter-tester agreement on DT was deemed good for inspiratory thickness (ICC=0.706), mod- erate for expiratory thickness (ICC=0.573) whereas SEM and SDD values were relatively small across testers. Inter- - 111 - Table 3. Diaphragm parameter measurements across nulliparous and parous women. Tester 1 Tester 2 Parameters Mean (SD) P-value Mean (SD) P-value Nulliparous Parous Nulliparous Parous Inspiratory thickness 0.36 (0.04) 0.40 (0.07) 0.061 0.35 (0.06) 0.45 (0.09) 0.01* Expiratory thickness 0.23 (0.05) 0.25 (0.06) 0.484 0.21 (0.05) 0.26 (0.08) 0.067 Thickening fraction 0.56 (0.22) 0.70 (0.39) 0.324 0.77 (0.49) 0.80 (0.34) 0.895 Excursion 4.93 (1.26) 4.33 (1.18) 0.288 4.55 (1.05) 3.95 (1.27) 0.265 SD, standard deviations. * Statistically significant differences between groups. Table 4. Intra-tester reliability results (n=20). Tester 1 Tester 2 Parameters ICC 95% CI SEM SDD ICC 95% CI SEM SDD Lower - Upper Lower - Upper Inspiratory thickness 0.511 0.100 - 0.773 0.045 0.124 0.685 0.461 - 0.847 0.055 0.152 Expiratory thickness 0.617 0.251 - 0.828 0.032 0.088 0.691 0.470 - 0.851 0.045 0.124 Thickening fraction 0.481 0.210 - 0.724 0.276 0.764 0.501 0.232 - 0.737 0.367 1.018 Excursion 0.756 0.558 - 0.888 0.662 1.834 0.700 0.482- 0.855 0.045 0.124 ICC, intraclass correlation coefficients; CI, confidence interval; SEM, standard error of measurement; SDD, smallest detectable difference. Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 tester agreement on DE was very good (ICC=0.748); ho- wever, SEM and SDD values yielded some measurement variability across testers (0.6cm and 1.67cm, respec- tively). Inter-tester agreement for TF measurements was poor (ICC=0.346), revealing substantial variability be- tween testers. Larger SEM and SDD values (0.3cm and 0.82cm, respectively) were also reported across testers. Discussion This study investigated intra-tester and inter-tester reli- ability of measuring DT and DE using RUSI among two novice examiners, following a standardized protocol, in a cohort of healthy women including both nulliparous and parous individuals. Actual findings by both testers were more or less in line with values reported in previous studies for DT and DE in healthy populations.25,27,30 The incorporation of nulliparous and parous women was in- tended to provide a foundational assessment for future re- search in postpartum populations and explore potential differences in diaphragm function related to childbirth. However, the sample lacked diversity in terms of BMI, as participants with a lower BMI (<30 kg/m2) were inten- tionally enrolled, aiming to minimize the challenges as- sociated with obtaining less clear US images which are often linked to higher BMI levels. This practice aligns with previous reliability studies.30,31,33 Both intra- and inter-tester reliability yielded overall sat- isfactory results across all parameters except for TF. Intra- tester reliability for DT measurements in both inspiratory and expiratory phases yielded good agreement for re- peated measurements by the same tester. However, DT measurements appeared to be somewhat lower than those reported in previous studies.25,26,28,30,31 SEM and SDD for intra-tester measurements aligned with previous studies,30 indicating a similar level of precision and error. For DE, both testers yielded very good agreements, consistent with prior research.29,30 Similar agreement patterns were reported in inter-tester reliability, with a slightly lower agreement for expiratory thickness. Nevertheless, despite lower agreement com- pared to previous studies,25,26,31 SEM and SDD values re- mained relatively low, indicating minimal errors in in- dividual measurements. Additionally, between-tester agreement for DE was close to excellent (ICC=0.748), similar to that reported by Scarlata et al.28,29 for quiet breathing excursion. The observed low agreement in TF, both within repeated measurements and between testers, aligns with previous findings,33 indicating difficulties in achieving consistent measurements. Regarding parous and nulliparous women, no significant differences were observed in all but one measurement. In- terestingly, inspiratory DT was marginally increased in parous women, reaching statistical significance with one tester, and yielding a close but non-significant p-value with the other one. In view of the small sample size, ho- wever, (only 9 parous women), this finding should be in- terpreted with caution. Additionally, as the study was not powered for between-group comparisons, these findings should be considered exploratory. Previous research has reported reduced DE in postpartum women with lumbo- pelvic pain, but no differences in DT, during a challenging postural task. Hence, future work should further explore these associations between parity and diaphragm function through larger, adequately powered samples. While this study provides valuable insights, it is essential to acknowledge certain issues that may influence the in- terpretation and generalizability of our findings. Firstly, the relatively small sample size and the use of only two testers represent a notable limitation. Despite sample size calculations and the reliability achieved in this study, fu- ture studies should incorporate larger samples and more examiners to strengthen the evidence. Another limitation is the lack of validity testing against an expert examiner, as this was not within this study’s scope. Future studies should consider including expert validation to provide a more comprehensive understanding of the accuracy and consistency of diaphragm measurements. The sample’s relatively lower BMI as previously men- tioned (due to difficulties in visualizing the diaphragm in individuals with higher BMI, particularly among novice sonographers) could also limit the study’s generalizability. - 112 - Table 5. Inter-tester reliability results. Parameter ICC 95% CI SEM SDD Lower Upper Inspiratory thickness 0.706 0.393 0.872 0.045 0.124 Expiratory thickness 0.573 0.186 0.806 0.045 0.124 Thickening fraction 0.346 -0.102 0.677 0.297 0.822 Excursion 0.748 0.466 0.892 0.600 1.665 ICC, intraclass correlation coefficients; CI, confidence interval; SEM, standard error of measurement; SDD, smallest detectable difference. Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 Consequently, these findings may not directly apply to higher BMI populations. However, the focus of this pre- liminary study was on healthy women within a specific demographic aimed at developing initial insights into the reliability of diaphragm measurements amongst novice clinicians. Our reliability may be slightly lower than studies involv- ing experienced sonographers,25,28,30,31 making our findings especially relevant however, to novice users employing US imaging for rehabilitation purposes. Additional train- ing is certainly essential to refine testers’ skills in dia- phragmatic US imaging, improving accuracy and reliability in DT and DE measurements. However, TF may not be a suitable assessment outcome among novice prac- titioners. This could be attributed to several factors like measurement variability, where small errors in static thick- ness measurements contribute to the variable dynamic cal- culation of TF, and breathing variability of the subject, where slight inconsistencies in breath depth affect DT. These findings suggest that TF values require more ad- vanced skills, being better suited for use by experienced sonographers or across highly standardized research set- tings. For novice practitioners, prioritizing simpler and more direct indicators, such as DE or static DT may pro- vide more reliable and clinically valuable information dur- ing training and early clinical application. Subject fatigue and the need for standardized subject ef- fort also require consideration. Subject fatigue was ob- served during the deep breathing phase, requiring three maximal inspiratory efforts (for DT measurement), as well as during quiet breathing (for DE measurements), requir- ing 1-3 respiratory cycles per captured image. This might have introduced some measurement variability, and it may have contributed to the slightly lower reliability in dia- phragm measurements performed by the testers. Although verbal instructions were consistently repeated across trials, further standardization of the method, perhaps by training subjects with an incentive spirometer as biofeedback to generate the same volume during each respiratory effort, as described by Orde et al.27, could enhance between-mea- surement and between-tester agreement. These findings hold significant implications for respira- tory muscle rehabilitation across diverse settings, includ- ing pregnant and postpartum women, athletes, and those with trunk dysfunctions, supporting the use of diaphrag- matic RUSI by physiotherapists, who typically have less professional experience in US imaging techniques com- pared to other clinicians.39 They show that diaphragmatic RUSI can be a quick and feasible assessment method that can also be effectively applied to patient populations. It should be noted that initially, capturing all six images re- quired approximately 30 minutes per tester. There was an observable learning curve; however, with increased ex- perience and practice, the time required decreased to ap- proximately 20 minutes. This progression indicates that the method is both feasible and efficient for quick clinical assessments. Additionally, the involvement of a consultant radiologist was initially necessary to ensure image quality, but her assistance became less needed as testers gained proficiency. This finding supports the potential for broader implementation in clinical and research settings, particu- larly for novice users. Thus, healthcare professionals unfamiliar with diaphrag- matic US measurements can become proficient through structured hands-on training and standardized protocols provided by experienced sonographers. Incorporating RUSI educational modules into formal academic curric- ula, whether in physiotherapy programs, Physical Re- habilitation Medicine (PRM) residency training, or through continuing professional education, with super- vised hands-on practice and performance evaluation under the supervision of US experts, could help bridge the gap between theoretical knowledge and clinical application, expanding diaphragmatic RUSI accessibility across var- ious clinical settings.11 Adherence to clearly defined pro- tocols can also ensure consistency, reduce variability, and accelerate skill acquisition. Furthermore, standardized procedures, especially during tidal breathing or full in- spiration/expiration maneuvers, can improve inter-tester reliability. As this study showed, even brief but well-struc- tured training programs can yield reliable results when standardized protocols are consistently applied. Understanding the reliability of DT and DE measure- ments, can shape therapeutic interventions and guide clin- ical decision-making. Research suggests that training respiratory muscles using tapered flow resistive or thres- hold loading devices may benefit spinal stabilization and improve symptoms such as low back pain.17,40,41 Stabiliza- tion exercises focused on inner unit muscles may also im- pact diaphragm thickness and mobility.42,43 DT and DE assessment using RUSI may thus contribute to an im- proved understanding of trunk muscle synergies and en- able the development of tailored rehabilitation interventions. Its potential applications may also contrib- ute to treating a wide range of conditions, including res- piratory, musculoskeletal, and urogynecological dysfunctions, providing clinicians and researchers with precise information about intervention efficacy. In post- partum care, where diaphragmatic breathing exercises are common, our study provides insights into the diaphragm’s role in stability. Thus, incorporating US assessments into postpartum rehabilitation can tailor interventions based on reliable measurements, enhancing overall trunk function. Conclusions This study provides valuable insights into the good intra- tester and inter-tester reliability obtained by novice ex- aminers in assessing diaphragm thickness and excursion with US across both nulliparous and parous healthy women. These findings add to the growing body of literature sup- porting the potential application of RUSI through measure- ments of diaphragm thickness and excursion in respiratory or musculoskeletal/postpartum rehabilitation and broader clinical contexts. They also contribute to establishing opti- mal practices and emphasize the significance of standard- ized protocols for meaningful and reliable results. Further research with larger and more diverse populations is needed to explore RUSI’s applicability in clinical conditions bey- ond this study’s scope. - 113 - Diaphragm measurement reliability in novice RUSI users Eur J Transl Myol 35 (4) 13907, 2025 doi: 10.4081/ejtm.2025.13907 List of abbreviations COPD, Chronic Obstructive Pulmonary Disease DE, Diaphragm Excursion DT, Diaphragm Thickness RUSI, Rehabilitative Ultrasound Imaging SDD, Smallest Detectable Difference SEM, Standard Error of Measurement TF, Thickening Fraction US, Ultrasound/Ultrasonography Contributions AS, EB, conception, writing - original draft preparation; AS, EB, MA, ED, EK, and IM, design, writing - review and editing; AS, EB, DTP and MT, Formal analysis and investigation; MT, EK and IM, resources; EB, ED and IM, supervision. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Publication charges were covered by the department of Physiotherapy, University of Patras. Conflict of interest The authors have no relevant financial or non-financial in- terests to disclose. Ethics approval and consent to participate The study was approved by the Ethics Committee of the University of Patras (number 15543/12-04-2023). All par- ticipants signed an informed consent form before partici- pating in the study. Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Conference presentation Some preliminary data of the study were presented in a poster at the 31st Annual Panhellenic Physiotherapy Con- gress, held at the War Museum, Athens on Dec 1-3, 2023. Corresponding author Anastasia Skoura, Laboratory of Clinical Physiotherapy and Research, Department of Physiotherapy, School of Health Rehabilitation Sciences, University of Patras, Building B, Central Campus, 26504, Rio-Patras, Achaia, Greece. Tel.: +30.2610.962406 ORCID ID: 0009-0007-4446-3970 E-mail: a.skoura@upatras.gr Co-authors Evdokia Billis ORCID ID: 0000-0002-5828-809X E-mail: billis@upatras.gr Maria Andriopoulou ORCID ID: 0000-0001-9570-3742 E-mail: mariandriop@yahoo.gr Elena Drakonaki ORCID ID: 0000-0002-3629-3277 E-mail: drakonaki@yahoo.gr Dimitra Tania Papanikolaou ORCID ID: 0000-0002-9453-5238 E-mail: dpapanikolaou@upatras.gr Maria Tsekoura ORCID ID: 0000-0003-2649-0522 E-mail: mariatsekoura@upatras.gr Eleni Kortianou ORCID ID: 0000-0003-2214-5545 E-mail: ekortianou@uth.gr Ioannis Maroulis ORCID ID: 0000-0002-6221-6099 E-mail: ioan.maroulis@gmail.com References 1. Hodges PW, Butler JE, McKenzie DK, Gandevia SC. Contraction of the human diaphragm during rapid pos- tural adjustments. J Physiol 1997;505:539–48. 2. Hodges PW. Is there a role for transversus abdominis in lumbo-pelvic stability? Man Ther 1999;4:74-86. 3. Hodges PW, Gandevia SC. Activation of the human diaphragm during a repetitive postural task. 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The effect of sta- bilization exercises on diaphragm muscle thickness and movement in women with low back pain. J Back Musculoskelet Rehabil 2018;31:323-29. Disclaimer All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af- filiated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 7 April 2025. Accepted: 25 July 2025. Early access: 27 October 2025. - 116 -