We determined the impacts of light intensity, blood fraction, fasting and storage time on glucose, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) concentrations in 140 broiler chickens assigned to two light intensities (5 or 20 lux/m²) and seven fasting times (0, 2, 4, 6, 8, 10, and 12 h). The collected blood was fractionated into serum or plasma and stored at -20°C for 0, 15, 30, 60, and 120 days. Serum glucose concentrations declined linearly by 1.15 mg dL-1 every 2 h of fasting, whereas plasma glucose responded quadratically (minimum point at 8 h and 13 min of fasting). Serum ALT concentrations showed a quadratic effect, with a maximum point at 3 h and 46 min of fasting. Serum GGT concentrations were not influenced by fasting time, whereas plasma decreased by 1.60 IU L-1 at each 2 h of fasting. The AST concentrations were influenced by fasting. Storage times influenced the glucose concentrations in a quadratic manner, with maximum points in serum and plasma at 95 and 72 days, respectively. Serum cholesterol and triglyceride concentrations increased linearly with storage time. Storage time caused a quadratic response on serum ALT, with a maximum point at 32 days, whereas there was a minimum point at 93 days in plasma. Concentrations of GGT increased linearly with storage. Glucose, triglycerides, AST, and ALT concentrations were highest at 20 lux. Samples for AST analysis can be stored up to 120 days at -20°C. Serum glucose, cholesterol, triglyceride, ALT, and GGT analysis is indicated with a 6 h fasting time, without freezing. The light intensity should be specified.