This study investigated "creep" in vancomycin and daptomycin MICs among methicillin-resistant Staphylococcus aureus (MRSA) isolates from blood cultures over a 5-year period in a hospital in the United Kingdom, using different susceptibility testing methods. Trends in vancomycin and daptomycin susceptibility were evaluated by using Etest performed prospectively on isolates in routine clinical practice from December 2007 to December 2010 (n = 102). Comparison was made to results from prospective testing of subcultures at the Scottish MRSA Reference Laboratory, using an automated system (Vitek 2) and retrospective testing (Etest and CLSI reference broth microdilution [BMD] method) of stored isolates from 2006 to 2010 (n = 208). Spearman's rank correlations revealed a significant increase in vancomycin MIC (P = 0.012) and a significant decrease in daptomycin MIC (P = 0.03) by year of study for Etest results from the time of isolation. However, neither trend was replicated in MICs from automated or retrospective testing. The Friedman test revealed a significant difference between vancomycin MICs obtained from the same samples by different testing methods (χ(2) [3 degrees of freedom] = 97; P < 0.001). MICs from automated testing and Etest analysis of stored isolates were significantly lower than those from Etest analysis at the time of isolation for both antibiotics (P < 0.001). Effects of storage on the MIC appeared within the first 6 months of storage. Inconsistent evidence on vancomycin MIC creep and the relevance of the MIC to clinical outcome may arise from differences in susceptibility testing methods, including storage of isolates. There is a need to standardize and streamline susceptibility testing of vancomycin against MRSA.