An experiment was conducted to study the relationship between behavioral and metabolic characteristics of acidosis challenged cows with their health. Eight Holstein cows in early lactation stage were allocated to four dietary treatments in a changeover design with factorial arrangement of 2´2 including two levels of alfalfa hay particle size (5 and 20mm theoretical length cut) and two levels of TMR dry matter (without and with adding water up to 50% DM). The results showed that ruminal pH measurements in cows were in safety margin, however some characteristics such as blood bicarbonate (21 vs 24 mg/dL), fecal particle size (4 vs 1.18 for fecal critical particle size), TMR sorting by cows (sorting for long hay from 19 mm sieve) and feed intake fluctuations indicated that the cows suffer from acidosis. In comparison with spot measurements of ruminal pH, metabolic and behavioral characteristics on animal can be used more efficiently for detection of acidosis challenged cows.