An experiment was carried out to investigate the effects of short-term fasting periods on the serum biochemical characteristics of rainbow trout, Oncorhynchus mykiss. For this purpose, fish were fasted 0, 6, 12, 24, 48 and 72 h before blood sampling. Thereafter the serum levels of thyroxine (T4), 3,5,3′-triiodothyronine (T3), cortisol, glucose, lactate, triglyceride, cholesterol, total protein, albumin, globulin and albumin : globulin ration (A : G) were determined. Results show that serum levels of T4 (4.60–8.77 ng ml−1), T3 (7.50–13.3 ng ml−1), cortisol (7.91–24.5 ng ml−1), glucose (18.5–80.1 mg dl−1), lactate (12.7–29.6 mg dl−1), triglyceride (171–500 mg dl−1), and cholesterol (321–535 mg dl−1) were significantly affected by the fasting period. However, there were no significant changes in serum total protein (3.03–3.68 g dl−1), albumin (1.78–2.01 g dl−1), globulin (1.15–1.70 g dl−1) or A : G (1.13–1.93) among the fish fasted 0–72 h. Results clearly show the importance of a fasting period on the serum biochemical properties of rainbow trout. According to the results, 24 h fasting is suggested as a pre-sampling fasting period in rainbow trout to measure serum levels of T4, T3, cortisol, glucose, lactate, triglyceride, and cholesterol. Potential mechanisms related to the changes in biochemical properties are discussed.