To characterize variability of various musculoskeletal biomarkers by different blood sampling techniques in conscious rats, plasma asparate aminotransferase (AST), creatine kinase (CK) and its isoenzymes, fatty acid binding protein 3 (FABP3), myosin light chain 3 (Myl3) and microRNA (miR-133a) obtained by jugular venipuncture (C-JV) or tail venipuncture (C-TV) were compared with those obtained by jugular venipuncture (A-JV) in isoflurane-anesthetized rats. Plasma CK, FABP3 and Myl3, especially when collected by C-TV, were higher with larger variability than when collected by A-JV, whereas miR-133a displayed large variability in all techniques. Interestingly, higher CK obtained by C-JV or C-TV was largely attributable to higher CK-MM or CK-BB, respectively. Handling and restraint stress were identified as possible factors contributing to larger variability for CK, FABP3 and Myl3. A close correlation between CK and FABP3 was demonstrated both in the C-JV and C-TV techniques. Next, we evaluated the impact of C-JV and C-TV techniques for detecting skeletal myopathy in 2,3,5,6-tetramethyl-p-phenylenediamine-treated rats. In this model, CK and CK-MM obtained by C-TV were significantly increased, but those obtained by C-JV were not modified. In contrast, AST, FABP3, Myl3 and miR-133a obtained by both techniques were drastically elevated to a similar extent. The results suggest that, in conscious rats, the tail venipuncture technique may be more appropriate to detect skeletal myopathy despite the higher variability with this technique than with the jugular venipuncture technique. Furthermore, FABP3, Myl3 and miR-133a may serve as more sensitive biomarkers with large signal-to-noise ratios regardless of the blood sampling technique in conscious rats.