Four experiments were conducted using mature Suffolk ewes to evaluate the effects of blood sample handling, processing and storage on measurements of the energy metabolites, β-hydroxybutyrate, total cholesterol, glucose, l-lactate, nonesterified fatty acid (NEFA), and triacylglycerol. In experiment 1 the effects of anticoagulants on metabolite measurements and packed-cell volume (PCV) were evaluated. Blood samples ( n=12) were collected into one of four evacuated blood-collecting tubes: no anticoagulant (SER; yields serum), and plasma-yielding sodium heparin (HEP), sodium fluoride and potassium oxalate (NAF), and tripotassium ethylenediaminetetraacetic acid (K 3EDTA). Anticoagulant affected ( P<0.05) metabolite values, with total cholesterol, triacylglycerol, and l-lactate highest in SER, and glucose highest in HEP; NEFA and β-hydroxybutyrate measurements were not affected ( P>0.10) by anticoagulant. In addition, among the plasma-yielding tubes, PCV was highest in HEP and lowest in NAF ( P<0.01). Experiment 2 investigated the effects of repetitive freezing-thawing cycles of plasma on metabolite levels. Blood samples ( n=20) were collected using NAF tubes, and plasma was stored at −20 °C and thawed in a water bath (21 °C) 0, 1, 2, and 3 times within 18 h after collection. Compared with fresh samples (Thaw 0), by thaw 2, l-lactate increased ( P<0.01) 5%, and glucose and total cholesterol decreased ( P<0.001) 13 and 4%, respectively. Plasma NEFA increased 10% ( P<0.01) between thaws 0 and 1, but returned to fresh levels (Thaw 0) with subsequent freeze-thaw cycles ( P<0.05). Plasma β-hydroxybutyrate decreased ( P<0.01) between thaws 0 and 1, but there was no further decline with subsequent freeze-thaw cycles ( P<0.05). Experiment 3 evaluated the effects of plasma storage temperature (−20 °C versus −80 °C) and length (0–180 days) on metabolite levels in blood samples ( n=12) collected in NAF tubes. All plasma metabolites were affected by storage length (day effect, P<0.01) but only total cholesterol values were affected by storage temperature, with values being higher in plasma stored at −20 than −80 °C ( P<0.01). Glucose measurements were higher in samples stored at −20 °C for up to 30 days, but were higher thereafter in samples stored at −80 °C (storage length × temperature effect, P<0.01). Experiment 4 evaluated the effects of anticoagulant (SER versus NAF) and whole blood storage (4 °C) for 0, 1, 3, and 7 days on metabolite concentrations. Glucose was higher ( P<0.0001) in NAF, possibly the result of the presence of the glycolytic inhibitor, sodium fluoride, whereas l-lactate, NEFA, total cholesterol and β-hydroxybutyrate were higher in SER ( P<0.01). Total cholesterol, l-lactate, and NEFA increased, whereas β-hydroxybutyrate decreased with days in storage ( P<0.01). Development of hemolysis in the samples artifactually elevated glucose and NEFA values by as much as 41 and 230%, respectively. Attention to proper blood handling, processing, and storage procedures, and avoidance of hemolysis are important in blood clinical analyses and in the proper interpretation of experimental results.