Introduction The erythrocyte sedimentation rate (ESR) includes three phases, each prone to different interferences. Due to many disadvantages of the reference Westergren method, modified and alternate methods have been introduced. The aim of this study was to compare the modified Westergren method on SRS 100/II analyzer in citrate blood with the alternate method on iSED® analyzer in EDTA sample. Additionally, possible interfering factors and ESR stability during 6 h at room temperature were evaluated. Methods A total of 188 samples were included in the method comparison. Additionally, the effects of inflammation, haematocrit and MCV values on ESR were evaluated. To determine ESR stability in different samples, ESR was evaluated at three time points; within 15 min of blood sampling and after 3 and 6 h in different sample types and analyzers (N = 65). Results Results indicated the constant difference between tested methods with obtained mean bias of 5 mm (95% CI: 3–7). There was higher absolute mean bias in groups with ESR > 40 mm and elevated inflammation markers (p < 0.001). Regarding different MCV and haematocrit groups there was no statistically significant difference in obtained absolute mean biases for MCV (p = 0.087) while there was higher bias in low haematocrit group compared to normal haematocrit (p = 0.004). In addition, there was a significant difference between ESR values at different time points for iSED® (p < 0.001) and no difference for SRS 100/II analyzer (p = 0.406). Conclusion There are differences in ESR values between tested methods. EDTA sample on iSED® should be analysed as soon as possible to avoid falsely increased ESR.