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Ultrasound imaging is widely used to measure architectural features of human skeletal muscles in vivo. We systematically reviewed studies of the reliability and validity of two-dimensional ultrasound measurement of muscle fascicle lengths or pennation angles in human skeletal muscles. A comprehensive search was conducted in June 2011. Thirty-six reliability studies and six validity studies met the inclusion criteria. Data from these studies indicate that ultrasound measurements of muscle fascicle lengths are reliable across a broad range of experimental conditions [intraclass correlation coefficient (ICC) and r values were always > 0.6, and coefficient of variation values were always < 10%]. The reliability of measurements of pennation angles is broadly similar (ICC and r values were always > 0.5 and coefficient of variation values were always < 14%). Data on validity are less extensive and probably less robust, but suggest that measurement of fascicle lengths and pennation angles are accurate (ICC > 0.7) under certain conditions, such as when large limb muscles are imaged in a relaxed state and the limb or joint remains stationary. Future studies on validity should consider ways to test for the validity of two-dimensional ultrasound imaging in contracted or moving muscles and the best method of probe alignment.

BackgroundSystematic reviews, which assess the risk of bias in included studies, are increasingly used to develop environmental hazard assessments and public health guidelines. These research areas typically rely on evidence from human observational studies of exposures, yet there are currently no universally accepted standards for assessing risk of bias in such studies. The risk of bias in non-randomised studies of exposures (ROBINS-E) tool has been developed by building upon tools for risk of bias assessment of randomised trials, diagnostic test accuracy studies and observational studies of interventions. This paper reports our experience with the application of the ROBINS-E tool.MethodsWe applied ROBINS-E to 74 exposure studies (60 cohort studies, 14 case-control studies) in 3 areas: environmental risk, dietary exposure and drug harm. All investigators provided written feedback, and we documented verbal discussion of the tool. We inductively and iteratively classified the feedback into 7 themes based on commonalities and differences until all the feedback was accounted for in the themes. We present a description of each theme.ResultsWe identified practical concerns with the premise that ROBINS-E is a structured comparison of the observational study being rated to the ‘ideal’ randomised controlled trial. ROBINS-E assesses 7 domains of bias, but relevant questions related to some critical sources of bias, such as exposure and funding source, are not assessed. ROBINS-E fails to discriminate between studies with a single risk of bias or multiple risks of bias. ROBINS-E is severely limited at determining whether confounders will bias study outcomes. The construct of co-exposures was difficult to distinguish from confounders. Applying ROBINS-E was time-consuming and confusing.ConclusionsOur experience suggests that the ROBINS-E tool does not meet the need for an international standard for evaluating human observational studies for questions of harm relevant to public and environmental health. We propose that a simpler tool, based on empirical evidence of bias, would provide accurate measures of risk of bias and is more likely to meet the needs of the environmental and public health community.

Non-technical summary Relaxed skeletal muscles behave like springs that resist joint motion.There have been few in vivo studies of the spring-like properties of relaxed muscles. In this study, ultrasound was used to image human calf muscles while muscle length was changed by rotating the ankle of relaxed subjects. The muscles of some subjects buckled at short lengths. At short lengths most muscle fascicles (bundles of muscle cells) are slack. As the muscle is lengthened the slack is progressively taken up, first in some fascicles then in others. The increase in muscle length is due partly to increases in the length of muscle fascicles but most of the increase in muscle length occurs in the tendons.Abstract Ultrasound imaging was used to measure the length of muscle fascicles in human gastrocnemius muscles while the muscle was passively lengthened and shortened by moving the ankle. In some subjects the muscle belly 'buckled' at short lengths. When the gastrocnemius muscle-tendon unit was passively lengthened from its shortest in vivo length by dorsiflexing the ankle, increases in muscle-tendon length were not initially accompanied by increases in muscle fascicle lengths (fascicle length remained constant), indicating muscle fascicles were slack at short muscle-tendon lengths. The muscle-tendon length at which slack is taken up differs among fascicles: some fascicles begin to lengthen at very short muscle-tendon lengths whereas other fascicles remain slack over a large range of muscle-tendon lengths. This suggests muscle fascicles are progressively 'recruited' and contribute sequentially to muscle-tendon stiffness during passive lengthening of the muscle-tendon unit. Even above their slack lengths muscle fascicles contribute only a small part (<∼30%) of the total change in muscle-tendon length. The contribution of muscle fascicles to muscle-tendon length increases with muscle length. The novelty of this work is that it reveals a previously unrecognised phenomenon (buckling at short lengths), posits a new mechanism of passive mechanical properties of muscle (recruitment of muscle fascicles), and confirms with high-resolution measurements that the passive compliance of human gastrocnemius muscle-tendon units is due largely to the tendon. It would be interesting to investigate if adaptations of passive properties of muscles are associated with changes in the distribution of muscle lengths at which fascicles fall slack.