In this work, we implemented an automated method using a correlation coefficient to select a time interval with a minimum movement or rest interval, together with analysis of variance for measurement of blood vessel diameter in the cremaster muscle. Video images binarization using analysis of variance resulted in an enhanced and a clearly defined vessel wall. Histamine (1 mM) induced a marked reduction in vascular diameter (vasoconstriction) in the cremaster muscle from mice fed with standard (SD) and high fat diet (HFD). However, the effect of histamine was reduced in HFD mice compared to SD mice. Thus, the change in vascular diameter was 87.14% ± 7.44% and 52.63% ± 16.27% in SD and HFD mice, respectively. In conclusion, determination of a rest interval with minimal movement and the use of analysis of variance resulted useful to evaluate vascular diameter in small arteries. We suggest this method to streamline experiments facilitating cardiovascular research.