Aim & Objectives: Intramuscular (IM) injections of 1 mL and 3 mL Diclofenac 75 mg are used in the clinic in the management of the pain. The present study was performed with the aim to evaluate muscle injury after IM injection of Diclofenac 75 mg/1 mL (Dynapar AQ ® , Troikaa Pharmaceuticals Limited, India) and Diclofenac injection 75 mg/3 mL (commercially available) in subjects using Magnetic Resonance Imaging (MRI) and laboratory blood tests. Material & Methods: Two separate single arm case studies, (each comprising of a single healthy subject) were carried out after Ethics Committee approvals and CTRI Registration. Two subjects who gave written informed consent received either Diclofenac injection 75 mg/1 mL (Dynapar AQ ® , Troikaa Pharmaceuticals Limited, India) OR Diclofenac injection 75 mg/3 mL (commercially available) and the muscle injury was evaluated at different time points up to 14 days. Results: In both the subjects, the intramuscular injury after the injection was reliably documented by MRI and laboratory tests. For the subject who received Diclofenac injection 75 mg/3 mL, there was significant muscle injury observed (T2- weighted magnetic resonance was increased manifold: the volume of muscle injury was 108 mL at 24 hr after injection; 137 mL at 48 hours, and progressive worsening after 48 hours, rising to 271 mL on Day 7 with subsequent normalization at Day 119). However, in the subject who received Diclofenac injection 75 mg/1 mL, muscle injury was relatively minor and reversed quickly at day 7; (T2-weighted magnetic resonance volume of muscle injury was 66 mL at 24 hr after injection, a flatter peak of 69 mL at 48 hours, declining thereafter). In the subject who received 3 mL injection, the CPK levels (Normal range: serum CPK: 39 – 308 U/L by immune assay) were highly increased (from baseline of 47 U/L to 1975 U/L in 8 hours, 1320 U/L at 48 hours, continued elevation after 48 hours: 420 U/L at 7 Days and 267 U/L on Day 119), with prolonged plateau of elevation and gradual decline over weeks indicating significant muscle injury with Diclofenac injection 75 mg/3 mL whereas, in the subjects who received diclofenac injection 75 mg/ 1 mL, there was only minor increase in CPK (from baseline of 93 U/L to 433 U/L in 24 hours) and was normalized to 95 U/L at day 7. In the subject who received Diclofenac injection 75 mg/3 mL, the IL-6 was within the upper range of normal (Normal Range: IL6 0-50 pg/mL by ELISA), and rose from a baseline of < 2 pg/mL to 37.3 pg/mL, but in the subject who received 1 mL IM Diclofenac, values stayed low throughout (<4 pg/mL). Conclusion: There was higher muscle injury following 3 mL Diclofenac intramuscular injection when compared to 1 mL Diclofenac intramuscular injection. This is the first comparative report confirming radiologic and laboratory evidence of muscle injury produced by a 1 mL and 3 mL Diclofenac injection, and opens the door for further research on muscle injury, and focuses our efforts to prevent or minimize the same.