The present trial reports the benefits of including within the habitual diet, different moderately high protein products (40% Carbohydrates-30% protein-30% lipids) for weight management, following a chronologically scheduled pattern. Thus it has been demonstrated that, within a free-living diet without dietary or energy restrictions, the substitution of a single meal by products with higher protein content resulted in a weight loss, due mainly to a fat mass reduction. In this sense, the results obtained with this work are in accordance with data previously reported [26, 28–31].
Ad libitum diets with high protein intake have been considered as useful approaches for effective weight loss and later maintenance [11, 28, 29]. Indeed, some long-term studies with no calorie restriction and programmed macronutrient distribution have resulted in a more effective weight loss and maintenance than conventional macronutrient distribution energy-restricted diets [11, 30, 31]. In a recent study, an isocaloric diet with a moderately high content in protein led to a body weight and fat mass reduction after 10 weeks , without affecting fat-free mass and maintaining glucose and lipid profile, which matches with the outcomes of the current intervention.
The short duration of the present trial together with a total modest increase of the total protein intake (from 18 to 22% of caloric intake) may have played a role on the slight, although statistically significant, weight and fat mass reduction (approximately 1 kg). However, a period of 4 weeks is a good predictor of body fat changes with a dietary approach [32, 33]. Indeed, when analyzing meals separately, the total protein content in breakfast, morning and afternoon snack has been increased in a range from 50 to 95% during the intervention compared to the free-living period, which seemed to be enough to produce a body weight reduction without caloric restriction, and gives support about the importance on timing of energy consumption, meal frequency and nutrient quality intake for weight maintenance [3, 4, 8, 11, 12, 34].
The combination of protein content and glycemic index in a diet could be determinant on body composition changes [11, 35]. In a recent study in Europe , it has been demonstrated the efficacy of high-protein, low-glycemic index diets on adults for weight maintenance . In this same study, it was suggested that the isolated effect of protein content or glycemic index in ad libitum diets did not influence body weight in children, while the conjunction of these two dietary factors has been shown as protective against obesity . Likewise, the current dietary intervention achieved an increase in protein intake together with a low glycemic index in the substituted meals, which may explain the observed improvement in body composition as assessed by bioimpedance measurements, a method which has been recently validated in our research group using Dual X-Ray Absorptiometry (DXA) as gold standard reference .
For the first time to our knowledge, it has been demonstrated that the inclusion of some specific meals in the habitual diet with high-protein low-glycemic index products may be sufficient for weight management, preserving lean mass and helping to decrease fat mass in type-2 diabetes patients, compared to a control period of free-living diet. These effects should be ascribed to the protein induced rise in thermogenesis [7, 11, 26, 36, 37], or even to the increased satiety consequence of high protein ingestion [38, 39], although the assessed satiety scores have not reflected this effect in the current study.
In relation to the lack of changes concerning glucose and insulin levels as well as the insulin resistance index, it may be due to the short intervention period, although previous studies with similar or even shorter periods have shown clinically relevant effects with respect to an improvement on insulin sensitivity .
Furthermore, contrasting with other nutritional intervention studies, our results did not show differences between the free-living and the intervention periods on lipid metabolism. Several studies have evidenced that exchanging protein for fat improves lipid-related cardiovascular risk profile [36, 40]. However, most of these studies used energy-restricted, low-fat, high-protein diets [37, 40–43]. Indeed, all the foods included in these hypocaloric diets were low fat products, decreasing also saturated fat intake, which could be one of the main factors involved in the reduction of total and LDL cholesterol. On the other hand and in agreement with our findings, several studies comparing high protein diets have not observed changes in lipid profile, concretely on total and LDL cholesterol [7, 37, 42]. However, a recent study by Morenga and co-workers  found that an ad libitum diet relatively high in protein improved total cholesterol and low-density lipoprotein cholesterol in comparison with standard dietary advice. In this case, the fiber content of the diet was also relatively high (>35 g per day), which could counteract the high fat consumption in this group . It is important to point out that, in the present study, the driven substitution of specific single meals by 40-30-30 products, led to a modest increase in the total dietary daily protein content (22%), which is lower than the quantities routinely used in high protein intervention programmes .
High protein diets have been also related with reductions in triglyceride levels [36, 37, 44, 45]. Comparing the duration of the present nutritional intervention period to other nutritional programmes, this one may have been relatively short to achieve significant changes in triglyceride levels. Indeed, triglyceride levels tended to be lower at the end of the nutritional intervention period in spite of not reaching statistical significance. Moreover, the effect on triglyceride levels has not always been observed in longer dietary interventions with a moderately high-protein content [7, 46]
A limitation encountered for the implementation of the current nutritional intervention has been the low sample size and the limited duration of the intervention, which can not permit us to generalize the outcomes obtained without further research. However, it is generally assumed that finding statistical significance with a small population is more difficult than when having a higher sample size. This outcome usually indicates that there is a real difference between the experimental periods. In any case, a type II error can not be discarded . Furthermore, as the study was designed as a longitudinal intervention with two consecutive periods, the first period is really a control, where the researchers only performed observational follow-up work. This approach has been already successfully employed and published elsewhere [48–51].
The overall results of this study may have been partly affected by the fact that the participants in the nutritional intervention were type-2 diabetes patients with initially controlled dietary treatment. In fact, the glycemic indexes in the breakfast, morning and afternoon snack during the free-living period were relatively low at baseline (< 55 units), and similar to the glycemic load reached with the products assayed. Thus, in another trial , the consumption of a low glycemic index breakfast during 21 days, compared to a high glycemic breakfast, led to a significant reduction on fasting glucose levels without affecting other biochemical biomarkers in obese subjects. In addition, a benefit on satiety was also reported, as it increased with the low glycemic index meal , which was not seen in the current trial. These observations suggest that in this population, the moment/time of consumption may be relevant in interpreting the results.
Moreover, when comparing two hypocaloric diets differing in the glycemic index, beneficial additional effects were found after weight loss (-5.3% vs. -7.5% change with the high- or low-glycemic diet, respectively) as well as in total- and LDL-cholesterol concentrations, where the decrease was 4-fold higher in the low glycemic index diet .
The reduction of glycemic index in a specific meal  or a diet  has also been associated to an increase in the satiety and a reduction on the voluntary food intake during the postprandial state. Indeed, the voluntary food intake may be an 80% higher after the consumption of a high glycemic vs. a low glycemic index meal . In this context, it is also in agreement with previous studies the similarity of the satiety scores observed between the free-living period and the intervention period in the present trial, as glycemic index remained unchanged between both periods.
The present results together with those from others [19, 34, 38] indicate the evident benefits of nutritional interventions on selected meals, giving an increasing importance to chrononutrition and meal frequency intake. Therefore, this is a good example of translational research carried out in a limited number of volunteers.