The Paleolithic (Paleo) diet is one modeled after the perceived food consumption of early human ancestors of the Paleolithic Era, consisting of mainly meat, fish, fruit, vegetables, eggs, and nuts. The purpose of this study was to examine the effects of a Paleo diet on blood lipids, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), non-HDL cholesterol, triglycerides (TG), total cholesterol (TC), and the ratio between TC and HDL (TC/HDL) in a healthy population. Healthy subjects of both genders (24 males, 20 females) were asked to eat an ad libitum Paleo diet for 10 weeks. Prior to the intervention, body weight, body fat percentage (BF%), maximal oxygen consumption (VO2max), TC, TG, HDL, and LDL were measured. These measurements were repeated following 10 weeks of a Paleo diet. As a whole, there was a significant increase in non-HDL (107.1±6.0 mg/dL to 120.2±6.5 mg/dL; P<0.01), LDL (93.1±5.4 mg/dL to 105.6±6.1 mg/dL; P<0.01), TC/HDL (3.0±0.2 to 3.3±0.2; P<0.05), and TC (168.8±5.4 mg/dL to 178.9±6.6 mg/dL; P<0.05) in healthy subjects following a Paleo diet. When stratified into groups based on initial blood lipid levels, deleterious changes were found in those with optimal HDL (82.1±3.2 mg/dL to 68.6±4.8 mg/dL; P<0.05), non-HDL (86.6±3.9 mg/dL to 101.4±4.8 mg/dL; P<0.01), TC (157.2±0.7 to 168.2±0.9 mg/dL; P<0.05), TC/HDL (2.5±0.1 to 2.7±0.1; P<0.05), and LDL (69.1±3.1 mg/dL to 83.5±4.1 mg/dL; P<0.01), whereas those within sub-optimal stratifications showed no significant changes. Subjects also decreased body weight (80.7±2.6 kg to 77.5±2.4 kg; P<0.001) and BF% (24.3±1.2% to 20.7±1.2%; P < 0.05). Our results demonstrate that an ad libitum unrestricted Paleo diet intervention is associated with deleterious changes to blood lipids in healthy subjects, despite concurrent improvements in body composition and cardiorespiratory fitness. Future research should focus on determining recommendations that embrace the positive aspects of the Paleo diet, while minimizing any deleterious impact on blood lipids in a healthy population.