**8. Conclusions**

We acknowledge the exciting potential and value of having a relatively inexpensive, portable, and non-invasive method to measure muscle glycogen in sports nutrition research and practice. Furthermore, we note that ultrasound techniques may provide new roles in sports nutrition, such as in the assessment of body composition. However, careful analysis of the literature, including previously unpublished results of our own study, fails to provide clear support for the use of an ultrasound technique (MuscleSound®) to measure muscle glycogen content or its changes due to supervised exercise and dietary treatments. This may be both a problem of the underlying principles of the technique, as well as the failure

of currently available algorithms to cover a larger range of changes in muscle glycogen, or other manipulations of muscle solutes and water, than are often seen in sports nutrition practice. We acknowledge that two validation studies have reported the apparent success of this technique in assessing changes in muscle glycogen in similar scenarios of diet and exercise. Notwithstanding these data, the validity of the use of this technique to assess muscle glycogen, especially in field uses where conditions and treatments may be less controlled then that achieved in research situations, must be considered equivocal. Further independent studies are warranted and should include a variety of scenarios in which muscle glycogen is manipulated across the range of concentrations commonly seen in athletes, with or without changes in muscle water and solute content. Interrogation of laboratory and real-world scenarios should also be included to investigate the tolerance of this method to differences in the logistics and rigour of data capture.

**Author Contributions:** Conceptualization, J.L.B., A.K.A.M. and L.M.B.; study methodology, J.L.B., M.L.R., K.A.T., N.A.J. and L.M.B.; statistical analysis, A.K.A.M. and J.L.B.; writing—original draft preparation, J.L.B., L.M.B. and A.K.A.M.; writing—review and editing, J.L.B., L.M.B., A.K.A.M., M.L.R., K.A.T. and N.A.J.; supervision, M.L.R. and L.M.B.; funding acquisition, L.M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Australian Catholic University ACU-FR Grant, awarded to L.M.B.

**Institutional Review Board Statement:** The study included in this review was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Australian Institute of Sport Human Research Ethics Committee (20140612) and the Australian Catholic University Human Research Ethics Committee (2014 254N).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study included in this review.

**Data Availability Statement:** The full data set reported in this study will be made available upon request to the corresponding author.

**Acknowledgments:** The authors would like to thank the athletes involved in the study, which produced the data included in this review, and the colleagues who contributed to its collection at the Australian Institute of Sport.

**Conflicts of Interest:** The authors declare no conflict of interest. MuscleSound® loaned the ultrasound equipment and provided results from their proprietary treatment of ultrasound images to produce the indirect estimates of glycogen storage reported in this review. However, they were not involved in the project funding, study design, data collection, data analyses and interpretation, the decision to publish the results or the preparation of this manuscript. The original data will be provided to MuscleSound® company upon the publication of this paper.
