PURPOSETo examine the validity of ultrasound (via cloud-based software that measures pixilation intensity according to a scale of 0–100) to noninvasively assess muscle glycogen in human skeletal muscle. METHODSIn study 1, 14 professional male rugby league players competed in an 80-min competitive rugby league game. In study 2 (in a randomized repeated measures design), 16 recreationally active males completed an exhaustive cycling protocol to deplete muscle glycogen followed by 36 h of HIGH or LOW carbohydrate intake (8 g·kg vs 3 g·kg body mass). In both studies, muscle biopsies and ultrasound scans were obtained from the vastus lateralis (at 50% of the muscle length) before and after match play in study 1 and at 36 h after glycogen depletion in study 2. RESULTSDespite match play reducing (P < 0.01) muscle glycogen concentration (pregame443 ± 65; postgame271 ± 94 mmol·kg dw, respectively) in study 1, there were no significant changes (P = 0.4) in ultrasound scores (pregame47 ± 6, postgame49 ± 7). In study 2, muscle glycogen concentration was significantly different (P < 0.01) between HIGH (531 ±129 mmol·kg dw) and LOW (252 ± 64 mmol·kg dw) yet there was no difference (P = 0.9) in corresponding ultrasound scores (HIGH56 ± 7, LOW54 ± 6). In both studies, no significant correlations (P > 0.05) were present between changes in muscle glycogen concentration and changes in ultrasound scores. CONCLUSIONSData demonstrate that ultrasound (as based on measures of pixilation intensity) is not valid to measure muscle glycogen status within the physiological range (i.e., 200–500 mmol·kg dw) that is applicable to exercise-induced muscle glycogen utilization and postexercise muscle glycogen resynthesis.