Subjects

Eligible subjects were 18–70 years old adults who gave written consent and had at least two of the following characteristics of the MetS [20]: 1. Central obesity (waist circumference ≥102 cm for men and ≥88 cm for women) 2. Elevated triglycerides ≥1.7 mmol/l 3. Reduced HDL-cholesterol <1.0 mmol/l for men and <1.3 mmol/l for women) 4. Raised BP ≥130/85 mmHg or BP medication and 5. Elevated fasting plasma glucose ≥5.6 mmol/l. Exclusion criteria were DM2, CVD, smoking, systolic BP > 180 mmHg, hypoglycaemic medication, pregnancy and severe internal or psychiatric disease. The study was approved by the Medical Ethics Committee Wageningen University and conducted according to the principles expressed in the Declaration of Helsinki.

Study settings

Subjects were recruited in September-October 2011 through advertisements in local newspapers and posters in primary health care units. After telephone contact a first selection was made and subjects were enrolled during a screening visit by the research physician. It was not known to the subjects whether any of the diets would be superior to the other (single-blinded). After randomization, subjects of the different diet groups could not communicate with each other. Before and directly after the intervention period subjects visited the Diakonessenhuis in Zeist for physical measurements, OGTT and blood sampling. All meals were delivered at their homes free of charge by a catering service.

Dietary interventions

The Palaeolithic-type diet intervention was based on anthropological Palaeolithic research [21, 22] with a concern for feasibility in modern times. It was based on lean meat, fish, fruit, leafy and cruciferous vegetables, root vegetables, eggs and nuts. Dairy products, cereal grains, legumes, refined fats, extra salt and sugar were not part of it. The reference diet was based on the guidelines for a healthy diet of the Dutch Health Council [23–31]. Both diets were designed as seven consecutive daily menus (breakfast, lunch, dinner and snacks) and provided on the basis of an isoenergetic intake of 8 700 kJ. Although coffee and tea were not part of a Palaeolithic-type diet, subjects were allowed to drink, in view of possible withdrawal symptoms, up to two cups of coffee or black tea per day. Any medication was continued at the same dosages. Additional detailed information on the two diets is summarized in Table 1.

Prevention of weight loss

Before the intervention all subjects recorded their usual diet using a food record diary. This diary was used by the dietician to adjust diets to individual energy demand by providing (additional) program-related snacks to prevent weight loss within the intervention period. Bodyweight was measured every second day. A daily bodyweight fluctuation of 2 kg was considered acceptable. Next to keeping bodyweight stable, importance was emphasized of keeping their usual exercise level during the intervention.

Compliance

To ensure compliance to the intervention, all subjects were requested to keep records of the food consumed. In addition, each subject was encouraged every other day by telephonic contact with their personal coach to complete all meals and to discuss their progress, bodyweight fluctuation, possible physical and psychological discomforts or adverse events. Palaeolithictype Diet and the Metabolic Syndrome Essay. A total of six personal coaches (each coaching 5–6 subjects) were involved during the study, who could be reached by the subjects for any questions concerning catering, daily recording or measurements.

Outcomes

Primary endpoints were glucose tolerance measured by the oral glucose tolerance test (OGTT) [32] and the characteristics of the MetS (waist circumference, systolic/diastolic BP, lipids, fasting glucose). Additional analyses comprised intestinal permeability, inflammation parameters (hsCRP, TNFα) and a stress parameter (salivary cortisol). Tolerability was measured by analysing the adverse events reported during the study and by performing blood tests for haematological indices and liver and kidney functions.

Measurements

Systolic and diastolic BP, from which the mean values were calculated, were measured twice after at least 10 min of sitting using an automated BP measuring device (OMRON M6 Comfort, OMRON Healthcare Co. Ltd.). Bodyweight was measured using a digital Seca 803 scale. For the OGTT subjects ingested a solution containing a 75 g glucose load. Plasma glucose and insulin concentrations were measured at 0 (baseline), 30, 60 and 120 min after ingestion. AUCs for glucose and insulin were calculated using the trapezoidal method. In addition HOMA_IR and the ratio between triglycerides and HDL-cholesterol were determined as measures of insulin sensitivity. Urine was collected for assessment of intestinal permeability, which was measured using the differential sugar absorption test (DSAT). After an overnight fast, the subjects ingested a test solution containing 5 g lactulose, 2 g mannitol and 5 g D-xylose in 100 ml water. All urine portions passed in the subsequent five hours were collected in a plastic container containing 0.5 ml 20% chlorhexidine as a preservative. Results were expressed as the urinary lactulose:mannitol ratio. Saliva was collected by Salivette® devices for assessment of the diurnal cortisol slope. Subjects had to chew on the cotton swab for 30–60 seconds. Sampling was performed at wake time, 30–40 minutes after waking (peak time), 17.00 h and bedtime [33]. A flattening slope of the diurnal cortisol curve is related to low-grade inflammation, insulin resistance and the MetS [34–36]. At the end of the intervention, an evaluation form on feasibility and motivational aspects was filled out by all subjects.

Baseline data

Tables 2, ​,33 and ​and44 show the baseline demographic and clinical characteristics of all subjects. Baseline data differed between both groups for BMI and number of characteristics of the MetS. No differences were found for coffee, tea and alcohol consumption, or the amount of exercise and stress factors between the intervention groups. All outcome variables exhibited normal distributions. Exception was the diurnal slope of cortisol showing a skewed distribution. Palaeolithictype Diet and the Metabolic Syndrome Essay.

Outcomes and estimation

Study subjects were 9 men and 25 women with an average age of 53.5 (SD9.7) years, a mean BMI of 31.8 (SD5.7) and an average number of 3.2 (SD1.3) characteristics of the MetS. Compared to reference, the Palaeolithic group, had lower mean systolic BP (−9.1 mmHg; P = 0.015), diastolic BP (−5.2 mmHg; P = 0.038), total cholesterol (−0.52 mmol/l; P = 0.037) and triglycerides (−0.89 mmol/l; P = 0.001) and a higher mean HDL-cholesterol (+0.15 mmol/l; P = 0.013). For detailed results see Tables 3 and ​and4.4. The number of characteristics of the MetS decreased upon the Palaeolithic-type diet (−1.07 characteristics; P = 0.010) compared to reference, resulting in less subjects with the actual MetS according to the definition of the MetS [20] after the intervention. There was a tendency for a larger decrease of AUC insulin in the Palaeolithic group (−1 918 mU/l x min versus +362 mU/l x min in the reference group; P = 0.051) using two-tailed unpaired t-tests, however this difference did not remain when adjusted for baseline BMI. In both groups change was observed in waist circumference (Palaeolithic: −3.1 cm; reference: −3.3 cm), fasting glucose (Palaeolithic: −0.9 mmol/l; reference: −0.35 mmol/l) and the ratio between urinary sodium and potassium (Palaeolithic: −1.1; reference: −0.57), however no differences were seen between both groups. Fasting plasma insulin (−2.7 mU/l) and HOMA_IR (−0.9) only decreased in the Palaeolithic group (Table 5). The ratio between total cholesterol and HDL-cholesterol (−1.16 mol/mol; P = 0.003) and the ratio between triglycerides and HDL-cholesterol (−0.91 mol/mol; P = 0.0001) were lowered in the Palaeolithic group compared to reference. Palaeolithictype Diet and the Metabolic Syndrome Essay. Among tolerability outcomes the white blood cell count and γ-glutamyltransferase showed a decline in favour of the Palaeolithic group. Urine analysis showed lower values of magnesium and calcium in the Palaeolithic group compared to reference. Other secondary outcome parameters showed no changes.

In line with the protocol, we took efforts to keep bodyweight stable. For nine subjects extra diet-related snacks were necessary due to over 2 kg weight loss, without being hungry (seven in the Palaeolithic group = 38% versus two in the reference group = 14%). Hunger was reported once by three subjects in the Palaeolithic group at the start of the intervention. According to food diaries and coach reports, all subjects were able to complete their dietary programs. At the end of the intervention bodyweight was nevertheless decreased in the Palaeolithic group compared to reference (−1.32 kg; P = 0.012). After adding this (unintended) weight loss as a separate fixed effect in a post-hoc analysis we found that favourable effects remained for systolic BP, HDL-cholesterol, triglycerides, the ratio between triglycerides and HDL-cholesterol, the ratio between total cholesterol and HDL-cholesterol and the number of characteristics of the MetS (Table 6). Palaeolithictype Diet and the Metabolic Syndrome Essay.

Interpretation

Our results are in line with results from earlier research in related target groups. We found a lowering of systolic and diastolic BP, where in uncontrolled studies with healthy volunteers [14, 15] as well as in controlled studies with diabetic patients [17] lowering of BP has also been reported. Contrarily, BP lowering was not observed in controlled studies concerning patients with ischaemic disease [16] and obese postmenopausal women [19]. With respect to lipids, we found positive changes in total cholesterol, triglycerides and HDL-cholesterol in favour of the Palaeolithic group. Previous research also reported positive changes in lipid profile in healthy subjects, diabetic patients or obese women [15, 17–19].Palaeolithictype Diet and the Metabolic Syndrome Essay. In our study, the ratio between total cholesterol and HDL-cholesterol, which is an important risk factor for CVD employed in clinical practice, was also lowered in the Palaeolithic group compared to reference. In addition, the ratio between triglycerides and HDL-cholesterol, a proxy for insulin resistance, was lowered in the Palaeolithic group compared to reference. Regarding glucose tolerance we did not find significant changes in our study. There was, however, a tendency for a larger decrease of AUC insulin and HOMA_IR in the Palaeolithic group. In contrast to our results, glucose tolerance improved within 10–17 days in healthy subjects after a Palaeolithic diet in an uncontrolled study [15], as well as in a controlled study of longer duration (3 months) in patients with ischemic heart disease [16]. In our study we found for both groups a decrease in abdominal circumference of approx. 3 cm, but not as a specific effect of the Palaeolithic-type diet. Earlier findings in 3 months [16, 17] and 6 months [19] controlled studies did report change in abdominal circumference in favour of the Palaeolithic-type diet, in patients with ischemic disease and DM2 [16, 17] or obese postmenopausal women [19]. Also other low-carbohydrate, high-protein or Mediterranean diets have demonstrated to be effective in improving various markers of cardiovascular risk in diabetic patients [37]. Unique for our study was the attempt to find these favourable effects independently from the factor of weight loss. Another notable observation was that characteristics of the MetS can be reduced within a short period in people with cardiovascular risk.

A frequently expressed critique on Palaeolithic-type diets is related to their low calcium content. In our study there was indeed a 50% lower calcium intake compared to the reference diet (Table 1). However, due to higher dietary magnesium intake compared to reference and compensation by lower urinary calcium and magnesium excretion, calcium homeostasis was unlikely to have become compromised.

Course of study and feasibility

Home catering with frequent coaching was a satisfactory method to monitor the intake and whereabouts of the subjects. Subjects remained motivated and were coached to consume the prescribed food. Problems could be solved adequately resulting in few drop-outs and missing data. Nevertheless, we experienced that delivering fresh foods in time across a wide region of the Netherlands was a logistical challenge. For follow-up studies, attention to catering aspects is of utmost importance. The experienced difficulties in maintaining bodyweight could be part of the positive effects of a Palaeolithic-type diet. Jönsson et al. [38, 39] reported that a Palaeolithic diet is more satiating per calorie than a Mediterranean-like diet. The composition of a diet is likely to be an important factor in satiety and bodyweight management. It has been reported that a high protein content of a diet can increase satiety and weight loss [40, 41]. What aspects of a Palaeolithic-type diet are important in this sense is yet unclear. Spreadbury et al.[42] reported that consuming a diet of grain-free whole foods may stimulate gastrointestinal microbiota more consistent with our evolutionary background. All of these factors might explain the apparent favourable effects of the modern “Palaeolithic” diets on satiety and metabolic health.

We like to thank the technicians of the laboratories of the Diakonessenhuis in Zeist and the University Medical Centre in Groningen for their work and commitment. We are grateful to Eric van Veluwe, chef-de-cuisine and caterer to show all of his best efforts to manage catering aspects and professor Leo Pruimboom and dr. Henk Huizing for their expert advise. This study was supported by funding from the Innovatienetwerk, Stichting Junio, the University Medical Centre Groningen and the University of Gerona.