Effects of lifetime food restriction on erythrocytes and numerous clinical chemistry, thyroid, parathyroid, and acid–base variables are reported from a paired-feeding study of 25% diet restriction in dogs. The 48 dogs were paired by gender and weaning weight within litter, and 1 dog in each pair was fed 25% less than its pair mate, from age 8 weeks until death. Erythrocyte and serum biochemistry profiles were evaluated by annual sampling intervals and longitudinally. Erythrocyte variables were slightly higher among control-fed dogs, a possible reflection of the need to support both higher body fat mass and lean mass that uses energy less efficiently. Among serum biochemistry variables, glucose and triglyceride were lower among diet-restricted dogs, while creatinine was slightly higher in the absence of renal disease or failure, over the life spans of the dogs. Glucose outcomes reflect improved glucose tolerance that has been demonstrated with diet restriction protocols in several species, while triglyceride data may reflect the difference in total body fat cells between feeding groups. Creatinine outcomes may reflect lean mass responses to diet restriction or more efficient function of the intracellular proteasome. Serum triiodothyronine levels were lower among diet-restricted dogs. Other clinical chemistry and thyroid variables, parathyroid variables, and acid–base variables were not strongly influenced by diet restriction but revealed age-related effects.