Purpose: To demonstrate the feasibility of using a susceptibility-based MRI technique with multi-echo gradient and spin echo (MEGSE) sequence to achieve simultaneous R2, R2' and R2* measurement and assess skeletal muscle oxygenation alternations in a rabbit model of unilateral artery embolization. Materials and Methods: Approved by the local institutional review board for experimental animal studies, nine New Zealand White rabbits were included in this study. The MEGSE sequence consists of embedding a set of gradient echoes around the echo of a single spin-echo sequence using several gradient echoes to collect the magnetization intensity during the formation and attenuation of spin-echo simultaneously after 180 degrees radio frequency pulse. Within-session and between-day tests were conducted to evaluate the reproducibility of this skeletal muscle oxygenation alternations measurement. Furthermore, all the MEGSE scans of skeletal muscle were conducted using a 3-T clinical MRI scanner during resting state (before unilateral artery embolization operation, pre), 1 h after unilateral artery embolization operation (post1) and 2 h after unilateral artery embolization operation (post2) model to verify the feasibility and sensitivity of this method. Results: The within-session coefficient of variations (CVs) of R2, R2' and R2* measurements were 1.57%, 3.33% and 2.57%, while the between-day CVs of were 1.42%, 5.85% and 2,85%. In all rabbits, the mean R2 decreased significantly from 36.46 +/- 1.03s(-1) (pre) to 30.58 +/- 2.11 s(-1) (post1, **P < 0.01, relative to pre) and 28.62 +/- 1.53 s(-1) (post2, **P < 0.01, relative to post1), and the mean R2' went up markedly from 9.88 +/- 2.14 s(-1) (pre) to 16.10 +/- 2.74s(-1) (post1, **P < 0.01) and 17.33 +/- 2.25 s(-1) (post2, **P < 0.05). The mean R2* increased from 43.27 +/- 3.75 s(-1) (pre) to 47.90 +/- 5.08s(-1) (post1, *P < 0.05) and to 48.04 +/- 4.42 s(-1) (post2, NS, P > 0.05). Conclusion: This study demonstrates the feasibility of simultaneous R2, R2' and R2* measurement method for the evaluation of skeletal muscle ischemia. Besides, this study indicates the sensitivity of the R2 and R2' compared with R2* and especially the necessity of R2 and R2' measurement for the further evaluation of skeletal muscle ischemia which always causes both edema and hypoxia in a rabbit model of unilateral artery embolization.