Interpretation and implications for future research and clinical practice
Bilateral Anterior Thigh Thickness exhibited the greatest variance in relation to both position and exercise, the greatest of which was the effect of sitting in a chair. Increases in BATT were exhibited when participants were sat in a chair as compared to measurements performed on the examination couch. This is consistent with previous studies, which have shown increased RF cross-sectional area in the seated position compared to supine . As it was not possible to view the entire VI in all participants in this position, the demonstrated effect is likely to be an underestimate and the true difference may be even greater. To a lesser degree, BATT also increased after exercise. The exercise protocol used in this study was more intensive than a typical exercise protocol that might be used in a frail or hospitalised population. However, even small increments in physical activity could be equivalently demanding in people with frailty or acute illness; for an older frail person this could be simply the demand of walking across a room and getting onto an examination couch. Bilateral Anterior Thigh Thickness has been shown to have excellent intra-rater, inter-rater variability when using the same protocol (i.e. repeated measures in the same position); we are confident that these changes relate to the effect of position and exercise. Additionally, validity of measurements was ensured by review of all by a second experienced sonographer, including correct orientation of the RF over the femur.
Importantly, the difference in BATT between the recumbent and sitting positions (+ 1.44 cm) was greater than differences that have been observed in clinical studies measuring changes in muscle quantity in hospitalised populations  i.e. highly clinically significant. This difference is also greater than the 95% confidence intervals for estimated mean BATT in all positions. It is important to consider that the differences in BATT do not relate to true differences in muscle quantity within these short time frames. Increased BATT in the seated position likely relates to contraction and shortening of the RF with combined knee and hip flexion, leading to a greater cross-sectional area; the RF inserts at both the hip and knee joint . As it is not possible to measure muscle volume with ultrasonography, this emphasises why standardisation of protocols is vitally important. Similarly, BATT increased after exercise, likely related to persistent contraction of the quadriceps muscles. During exercise, metabolic requirements of skeletal muscles are increased and blood flow increases . This in turn increases the temperature of muscles and reduces stiffness, promoting increased muscle activity i.e. muscle contraction in the neutral position.
Echogenicity declined in the seated position, but there were no significant changes after exercise. Additionally, the change in the seated position was smaller and potentially of less clinical significance. Echogenicity provides a numerical measure of muscle quality, which has been shown to correlate with muscle function . Therefore, echogenicity may provide a more readily standardisable measure across settings, where standardisation of exercise protocols is challenging. However, echogenicity has been shown to exhibit greater inter-user variability compared to BATT . As all images for individual participants were obtained by the same sonographer, this should not have affected changes demonstrated across repeated measures for individual participants.
As much as possible, position should be standardised when performing quadriceps muscle ultrasonography; where there are any deviations in position, these should be noted. The seated position may represent an option as a pragmatic, easily standardised position. However, as we were unable to obtain thickness measurements in all patients in this position, this may be less feasible without readily available machines/probes that measure to greater depth. This is important when measuring healthy young adults as part of a reference standard, but may also be particularly relevant in individuals with increased subcutaneous tissue e.g. sarcopenic obesity. We recommend that ultrasonography measures should be taken with the knee in natural relaxation. As we did not find any clinically or statistically significant difference between the supine and 45o positions, small variations in the tilt of the head of the bed can be tolerated, provided significant flexion of the knee is avoided.
Less variance was exhibited with BIA. Phase angle, SMM-Janssen, fat percentage, TBW, ECW, and ICW did not vary across any repeated measures statistically significantly. There were reductions in SMM-Sergi from the 45o position to fully supine and after exercise. Pragmatically, this means that BIA can be performed in a variety of clinical settings, including where it is not practical to perform supine e.g. in a frail older person attending a clinic appointment in a wheelchair. A more reliable formula where the position of the upper body cannot be standardised but the patient/participant is able to lie on a couch or a period of rest prior to assessment is not feasible may be SMM-Janssen. Historically, BIA has been extensively criticised previously compared to DXA, CT or MRI in research settings, due to reduced precision . However, it is also important to consider the purpose of measuring muscle quantity and the degree of certainty that is necessary in clinical practice. BIA may be a pragmatic tool for screening and as an adjunct as part of a Comprehensive Geriatric Assessment. As well as less variability demonstrated in this study with positions and exercise, BIA is also much quicker to perform than ultrasound and requires minimal training. The phase angle has been proposed as a measure of muscle quality, as a measure of cell membrane function . However, BIA is known to be affected by fluid balance , although as technology and datasets develop it may be possible to perform correction calculations for this. BIA is also currently contraindicated in people with implanted cardiac devices; there is increasing evidence that it is likely to be safe , but it is unknown if results can be reliably interpreted.
What are the limitations of this research?
Importantly, this research was performed in healthy young volunteers. Whilst our results provide preliminary results towards standardisation of a protocol for muscle quantity assessment, we recognise that results may be different in an older and/or hospitalised population. In older adults with sarcopenia, less variability in measures may be seen if muscles are already very small and insufficient. Indeed, a pragmatic interpretation may be that if muscle quantity is demonstrated to be reduced in the seated position, then it is very likely to be reduced in any other position. However, if muscle quantity appears normal it may still be reduced if measured without the hip and knee in combined flexion.
Conversely, in hospitalised populations it is plausible that greater variability in measures may be exhibited due to greater fluid shifts. This may affect measurements taken using ultrasonography as well as BIA. In our study, all participants were young, healthy, and clinically euvolaemic. There was no clinical evidence of change in hydration status between repeated measures, and hydration status measured by BIA itself also did not change with position. Additionally, nearly all participants were sufficiently physically active to meet the minimum World Health Organization (WHO) guidelines, which may have affected the responsiveness of skeletal muscles to the effects of position and exercise. Our study was not powered to examine differences of position and exercise effect between groups (e.g. gender, ethnicity, activity levels). However, since participant characteristics did not change between repeated assessments, this will not have affected our overall results.
Whilst we consider the changes in BATT and BATT:SCR not to be related to true changes in muscle quantity, we recognise that we did not measure muscle quantity using any gold standard techniques. Due to the nature of the study, it was also not possible to blind assessors to position. Additionally, considering the effects of exercise, this study only evaluated the effects of very short high intensity exercise; the effects of longer periods of exercise, or less intensive physical activity are unknown. We also acknowledge that we cannot rule out effects of moving between positions in the order used, as we did not use a counterbalance design.