Our pre-agricultural, hunter-gatherer human ancestors during the Paleolithic period (the old stone age; 2.5 million – 10,000 years BP) had a diet based on vegetables, fruit, nuts, roots, meat, organ meats and insects 1. This Paleolithic diet has been suggested to be more in concordance with our evolutionary legacy than a diet based on products associated with agriculture during the Neolithic period (10,000 years BP – present time) such as cereals and milk 23. A diet in less concordance with our evolutionary legacy might confer diseases due to insufficient adaptation 4 possibly explaining the reported lower incidence of diseases of affluence 5, such as type 2 diabetes, obesity and cardiovascular disease among Non-Western ethnic groups with hunter-gatherer lifestyles and diets 67. However, the mechanisms behind this reported lower incidence of diseases of affluence are largely unknown, and commonly discussed dietary factors such as fat (amount and quality) and (cereal) fiber may be of limited importance 8910. A possible mechanism might be the potential of Paleolithic diet to reduce risk factors for diseases of affluence, such as disturbed glucose homeostasis, insulin resistance and hypertension 2. Food restriction induced by the satiating properties of a Paleolithic diet 2 may also play a role. Food restriction, sometimes referred to as dietary restriction or caloric restriction 11, of 20–40% of dietary energy compared to free feeding (ad libitum) has been shown to reduce the incidence of several diseases of affluence in mammals including non-human primates, and possibly humans 1213. Interestingly, Non-Western ethnic groups with hunter-gatherer lifestyles and diets stay lean and apparently reap health benefits similar to those induced by food restriction despite ad libitum availability of food 14.

The aim of this study was to determine experimentally whether a Paleolithic diet on a long-term basis affects risk factors for diseases of affluence in a prospective and randomized setting. This was assessed in domestic swine by examining glucose tolerance, post-challenge insulin response, plasma C-reactive protein and blood pressure after 15 months on a Paleolithic diet in comparison with a cereal based swine feed supplemented with rapeseed oil.

Energy intake at age 17 months was approximately 20% lower in the Paleolithic group despite much larger feed rations in terms of both volume and weight (Table 1). The mean weight curves for the two groups started to diverge after 3 months of feeding the different diets (Figure 1). At the end of the study the Paleolithic group weighed 22% less (129 ± 16 kg vs. 166 ± 28 kg, Paleolithic vs. Cereal, p = 0.0009; Table 2 and insert Figure 1) and was 6% shorter than the Cereal group (p = 0.003), while subcutaneous fat thickness was 43% lower (p = 0.0003; Table 2). No significant difference was seen in body temperature between groups (Table 2). The geometric mean of C-reactive protein was 82% lower in the Paleolithic group (p = 0.0007; Table 2). Intra-arterial diastolic blood pressure was significantly lower by 13% (p = 0.007) in the Paleolithic group, while systolic blood pressure was non-significantly lower by 7% (p = 0.12; Table 2).

No significant difference was seen in mean fasting values of glucose or insulin between the groups (Table 3). Parameters related to the overall metabolic status indicated unchanged total glucose AUC (p = 0.14; Table 3, Figure 2) and glucose disappearance rate (K_G = 0.58 ± 0.12 vs. 0.67 ± 0.17 %min^-1, Paleolithic vs. Cereal, p = 0.20; Table 3). Fasting insulin sensitivity was not affected by diet (QUICKI = 0.66 ± 0.15 vs. 0.70 ± 0.36, Paleolithic vs. Cereal, p = 0.7; Table 3), but dynamic insulin sensitivity was markedly higher in the Paleolithic group (dynamic insulin sensitivity = 2.35 ± 0.76 vs. 1.41 ± 0.39 %min^-1/(pmol/l), Paleolithic vs. control, p = 0.004; Table 3). Significant differences were also found in the insulin response to injected glucose (Table 3, Figure 3). AUCins_0–120 was significantly lower in the Paleolithic group by 47% (p = 0.001) and stimulated insulin secretion was even more reduced by 58% (p = 0.0005). This reduction was mostly ascribed to the second phase insulin secretion, since the acute insulin response immediately following the glucose bolus (indicator of the early phase insulin release) was not different between the two groups (14.9 ± 8.3 vs. 15.8 ± 10.7 pmol/l, p = 0.8, Paleolithic vs. control; Table 3).

Evaluations of bivariate correlation showed that weight was highly correlated with subcutaneous fat thickness (adjusted R² = 0.77, p < 0.0001). Evaluations of multivariate correlation with forward stepwise linear regression analysis was performed in order to study variables independently explaining the variation of dynamic insulin sensitivity, AUCins_0–120, log C-reactive protein and diastolic blood pressure, respectively. In each of these four regression models diet was entered as independent variable and evaluated with each one of the remaining relevant study variables as the second independent variable. Diet emerged as the strongest explanatory variable for variations in dynamic insulin sensitivity, AUCins_0–120, log C-reactive protein and diastolic blood pressure.

Immunohistochemical analysis comprised islet staining of insulin, glucagon, somatostatin and pancreatic polypeptide. There was no obvious difference between islets in the Cereal and Paleolithic groups regarding the frequencies or intra-islet distribution patterns of cells storing each of these four hormones, nor was the islet size or islet frequency overtly different (Figure 4). However, leukocytes, as revealed by their unspecific binding of the secondary antibodies, their small size, and their characteristic nuclei, were clearly more frequent (more than doubled by simple cell counting) in all the sampled pigs from the Cereal group as compared to all the sampled pigs from the Paleolithic group, with no overlapping between groups. These cells usually occurred scattered throughout the exocrine pancreatic parenchyma or clustered around pancreatic ducts and blood vessels (Figure 5).

This study showed highly beneficial effects of a Paleolithic diet on risk factors for diseases of affluence when compared to cereal based swine feed. A strength of the study is that the model used is one of the best non-primate models for human disorders, the domestic pig 20. The present study indicated no difference in glucose disappearance during IVGTT between the groups. During IVGTT plasma insulin increased successively between 30 and 80 min in both groups, possibly due to propofol anesthesia 2122, and this precluded the use of the minimal model of glucose disappearance 1617. However, other indices of dynamic insulin sensitivity clearly showed higher insulin sensitivity in the Paleolithic group, which also exhibited a significantly lower insulin response to injected glucose. It is worth noting that the difference in insulin response between groups during IVGTT was observed in the late phase of insulin secretion. Evaluation of multivariate correlation showed that the beneficial effect of Paleolithic diet on insulin sensitivity and insulin response was independent of all other relevant study variables such as body weight, subcutaneous fat thickness and body temperature. A Paleolithic diet thus conferred higher insulin sensitivity, which is central to the prevention of cardiovascular disorders 23, and consistent with our finding in humans 24. Furthermore, contrary to our survey in humans 24, this finding of higher insulin sensitivity was associated with no significant difference between groups in fasting levels of insulin and glucose, indicating that the Paleolithic diet affected insulin action mainly at the level of muscle and adipose tissues rather than the liver 18.

The Paleolithic group also showed significantly lower levels of C-reactive protein, a physiologic marker of subclinical inflammation, which has been shown to be associated with insulin resistance and cardiovascular disease 2325. Elevated levels of C-reactive protein has been suggested to reflect overproduction by expanded adipose tissue mass 23. However, evaluation of multivariate correlation showed that the beneficial effect of Paleolithic diet on C-reactive protein was independent of all other relevant study variables including measures of obesity such as body weight and subcutaneous fat thickness. Interestingly, immunohistochemical analysis suggested a diffuse and low-grade pancreatic inflammation in the cereal group, as evidenced by clearly more frequent leukocytes scattered throughout their exocrine pancreatic parenchyma or clustered around pancreatic ducts and blood vessels. This finding offers a novel approach in the research on the association between inflammation and type 2 diabetes 25. Immunohistochemical analysis also showed that the cereal group could compensate for both pancreatic inflammation and increased need of insulin due to lower insulin sensitivity, without noticeable differences in pancreatic islets between groups.

The shorter length in the Paleolithic group is not unexpected if we consider length in pigs as a correlate of height in humans. Available evidence suggests that hunter-gatherers and similar ethnic groups are shorter than Western populations, and a positive relationship between height and cardiovascular disease has been noted in international comparisons 26. Insulin resistance and hyperinsulinemia can hypothetically promote growth by way of insulin-like growth factors 27.

In addition to these significant results, intra-arterial diastolic blood pressure was significantly lower in the Paleolithic group, and this beneficial effect of Paleolithic diet was also independent of all other relevant study variables. The present study thus provides experimental evidence for beneficial effects of a Paleolithic diet on risk factors for diseases of affluence, which might account for their low incidence reported among Non-Western ethnic groups with hunter-gatherer lifestyles and diets 67.

In conclusion, this study in domestic pigs suggests that a Paleolithic diet as compared to a cereal based diet conferred higher insulin sensitivity, lower C-reactive protein and lower blood pressure. The results suggest that pigs, as previously suggested for humans 3, are not specifically adapted through evolution to a diet incorporating large amounts of cereals, which could confer diseases of affluence as a sign of insufficient evolutionary adaptation 4.

The author(s) declare that they have no competing interests.

TJ participated in the design of the study and in carrying out the experiments at the end of the study, participated in statistical analysis, and conceived of and wrote the article. BA participated in the design of the study, carried out the analysis of glucose and insulin, and conceived of and participated in the design of the article as well as revising it for important intellectual content. GP participated in the statistical analysis of glucose and insulin and revised the article for important intellectual content. FS and NW participated in carrying out the experiments at the end of the study, and carried out the immunohistochemical studies. SS and TS participated in the overall design and coordination of the study, were responsible for the animals and the laboratory where the experiment was performed, and participated in carrying out the experiments at the end of the study. MU participated in carrying out the experiments at the end of the study and revised the article for important intellectual content. JF carried out the analysis on C-reactive protein and revised the article for important intellectual content. LG revised the article for important intellectual content. SL conceived of and participated in the design, coordination and execution of the study, participated in carrying out the experiments at the end of the study, participated in statistical analysis, conceived of and participated in the design of the article as well as revising it for important intellectual content. All authors read and approved the final manuscript.