Table 3.

Protein intake and outcome BMI, growth, body composition, IGFS-I (6 clinical trials, 12 cohort, 5 cross-sectional)

Author, year (ref no.) Country Study design (study name if applicable)	No. of participants	Exposure (incl age)	Outcome (incl age)	Effect/association	Study quality comments
Budek, 2007, (14) Denmark, Cross-sectional	84 out of 96 boys (84%)	Intake of total, dairy and meat protein at 8 years	Concentrations of sIGF-I and markers for bone-turnover; serum osteocalcin (s-OC), bone-specific alkaline phosphatase (s-BAP) and C-terminal telopeptides of type l collagen (s-CTX) at 8 years	Dairy protein was negatively associated with sOC (p=0.05) but not significantly associated with sBAP and sCTX. Dairy protein decreased (p=0.05) sOC at a high meat protein intake (>0.8 g/kg), whereas meat protein increased (p=0.03) sOC at a low dairy protein intake (<0.4 g/kg). Total and meat protein intake was positively associated with sBAP (p≤0.04) but not significantly associated with sOC and sCTX. Free sIGF-I was positively associated with total (p<0.01) and dairy (p=0.06) protein but not with meat protein.	B Results not adjusted for mis/underreporting, no power calculation. Definition of meat protein includes meat, poultry and fish (but not pork, egg)
Dorosty, 2000, (32) UK Prospective cohort (ALSPAC)	772 out of 889 (87%)	Protein intake at 18 months (g/day and E%)	Timing of adiposity rebound (AR) (hypothesis: high protein intake promotes early AR)	No evidence of associations between protein intake, or any other dietary variable, and timing of the AR. Children with AR very early (≤ 43 months) or early (from 49 but before 61 months) had parents with sig higher BMI and were sig more likely to have at least 1 obese parent.	B Measurement errors in dietary recording not considered, very little data given about recordings, power calculation not done,
Gunnarsdottir 2003, (3) Iceland Nationwide longitudinal cohort	90 children (41 boys)	Size at birth, growth and food intake in infancy	BMI at 6 years (Weight and height were measured at maternity wards and healthcare centers in Iceland throughout infancy and at 6 years)	Weight gain from birth to 12 months as a ratio of birth weight was positively related to BMI at the age of 6 years in both genders (β = 2.9±1.0, p=0.008, and β = 2.0±0.9, p=0.032 for boys and girls, respectively). Boys in the highest quartile of protein intake (E%) at the age of 9–12 months had significantly higher BMI (17.8±2.4 kg/m2) at 6 years than the lowest (15.6±1.0 kg/m2, p=0.039) and the second lowest (15.3±0.8 kg/m2, p=0.01) quartile. Energy intake was not different between groups. Together, weight gain at 0–12 months and protein intake at 9–12 months explained 50% of the variance in BMI among 6-year-old boys. Rapid growth during the first year of life is associated with increased BMI at the age of 6 years in both genders. In boys, high intake of protein in infancy could also contribute to childhood obesity.	B Measurement errors in dietary recording not considered, power calculation not done
Günther, 2006 (33) Germany Cohort study DONALD	313 children with complete data (161 boys, 152 girls) up to 7 years	Habitual energy adjusted protein intake (E% and g/kg RBW/day, average between 2–3 dietary records between 12 and 24 months. RBW = reference body weight (adjusted for age- and sex-specific)	Timing of adiposity rebound (AR) (hypothesis: high protein intake promotes early AR and higher BMI at AR)	After adjusting for potential confounders, girls in the highest tertile (T3) of habitual energy-adjusted protein intake had a significantly higher BMI-SDS at AR than those in T1 (T1:-0.61 (95% CI: -0.90; -0.31), T2:-0.49 (-0.79; -0.20), T3:-0.08 (-0.36; 0.20), p for difference = 0.01). A comparable association existed with habitual protein intake expressed as g/kg RBW/day (T1:-0.64 (-0.93; -0.36), T2:-0.22 (-0.52; 0.09), T3: -0.25 (-0.54; 0.04), p=0.04). In boys, there were no differences in BMI-SDS at AR between tertiles of habitual protein intake (% of energy or g/kg RBW/day) (P40.05). Boys in the lowest tertile of habitual energy-adjusted protein intake tended to experience a later AR (T1: 6.0 (5.6; 6.4), T2: 5.5 (5.1; 5.9), T3: 5.4 (5.0; 5.9) years, p=0.07). But neither in girls nor in boys was age at AR significantly different between tertiles of habitual protein intake (% of energy or g/kg RBW/day) (p>0.05). A higher habitual protein intake between the age of 12 and 24 months was associated with a higher BMI-SDS at AR in girls, but not in boys. There was no consistent relation between habitual protein intake in early childhood and timing of AR.	B Measurement errors in dietary recording not considered, power calculation not done
Günther, 2007 (34) Germany Prospective cohort, DONALD Study	203 (104 M, 99 F)	Protein intake at 6, 12 and 18–24 months	BMI and %BF (per cent body fat) at 7 years of age	↑protein →↑BMI Consistently high protein intake 12, 18–24 months positively related to increased BMI SDS and %BF at 7 years; BMI SDS 0.37 (95% CI 0.12. 0.61) vs. 0.08 (-0.09, 0.26), p=0.04; %BF 18.37 (17.29, 1.51) vs. 16.91 (16.19,17.66), p=0.01. OR for BMI >75th percentile 2.39 (1.14, 4.99), p=0.02; OR for %BF >75th percentile 2.28 (1.06, 4.88), p=0.03. No effect of protein intake at 6 months.	A Reported energy intake a bit low in one group (<−20% of standard for age for the low-low group, Table 2)
Günther, 2007 (35) Germany Prospective cohort, DONALD Study	203 (102 M, 101 F)	Protein intake at 6, 12, 18–24 months, 3–4 years, 5–6 years	BMI and %BF (per cent body fat) at 7 years of age	12 months and 5–6 years identified as critical periods at which higher total and animal, but not vegetable, protein intakes were positively related to body fatness at 7 years. Animal protein intake at 12 months positively associated with %BF [mean/tertil [95% CI) T1; 16.2(15.23,17–25), T2; 17.21(16.24, 18.23), T3; 18.21(17.12, 19.15), p=0.008 Animal protein E% at 12 months positive association to BMI a 7 years (p=0.02) Animal protein E% at 12 months and 5–6 years positive association to %BF at 7 years (p=0.01). Dairy E% at 12 months, but not meat or cereal, positive association to BMI a 7 years (p=0.02) and %BF at 7 years (p=0.07).	A 2.6% records excluded (potentionally implausible FIL/PAL) Median energy intake just above <20% so credible but a bit low.
Hoppe, 2009, (27) Denmark CT	57 boys	At 8 years a 7-day intervention with 540 ml milk-based drinks, either: 1) whey with low mineral content (Ca and P) (Whey-low), 2) whey with high mineral content (Whey-high), 3) casein with low mineral content (Case-low), 4) casein with high mineral content (Case-high)	Serum IGF-1, IGFBP, fasting insulin, C-peptide, index of insulin resistance, glucose	No interactions between milk mineral groups (high, low) and milk protein groups (whey, casein). The milk protein intervention groups were combined. Average daily protein intake was increased by 17% by the whey drink, from 58 g/day (2.23 g/kg per day, 12.98 PE%) to 68 g/day (2.56 g/kg per day, 15.42 PE%) (p<0.001), and by 51% by the casein drink, from 68 g per day (2.30 g/kg per day, 14.30 PE%) to 103 g per day (3.44 g/kg per day, 23.40 PE%) (p<0.001). In the whey group, fasting insulin increased by 21% (p=0.006), with no change in IGF-1 (p=0.27). In the casein group, serum IGF-1 increased by 15% (p<0.0001), whereas there was no change in fasting insulin (p=0.36).No independent effects of a high milk mineral intake on IGF-1 and insulin. Increase in serum urea nitrogen (SUN), and the molar ratio of IGF-1/IGFBP-3 was significantly higher in the combined casein-group than in the combined whey group. Conversely, whey increased fasting insulin more than did casein.	B 36% drop-out. No details given. Remaining diet unclear. Energy intake at baseline reported and credible level. Nothing said about 7-day Measurement errors not considered Can′t find that they say very much about compliance.
Hoppe, 2004 (28) Denmark Intervention study (7-day)	24 boys	At 8 years a 7-day intervention with 53 g protein daily, 12 boys as 1.5 l skimmed milk, and 12 boys as 250 g low fat meat. In addition, they were asked to eat their normal diet ad libitum.	IGF-I concentrations and the molar ratio of IGF-I/IGFBP-3 in healthy, prepubertal children	After 7 days, the average protein intake increased in milk group by 61%; meat group +54%. The milk group increased s-IGF-I by 19% (p=0.001) an s-IGF-I/s-IGFBP-3 by 13% (p<0.0001). No increase in the meat group. Conclusion: Compounds in milk and not a high protein intake as such seem to stimulate IGF-I. This might explains the positive effect of milk intake on growth seen in some studies.	B No power calculation Compliance is not reported. It is stated that protein intake is increased but nothing about how much of the additional 15 dl of milk/250 g of meat was actually taken, and very little about how their habitual diet changed.
Hoppe, 2004 (36) Denmark Prospective, observational cohort	142 with data from 9 months invited to 10 years follow-up, 105 (74% agreed to take part), 51 M and 53 F (+ 1?)	Protein intake at 9 months, and 10 years Protein intake (as measured by SUN (serum urea nitrogen) and IGF-I at 10 years	Weight and height at 10 years	SUN at 9 months was a predictor for weight at 10 years: 0.96 (0.28–1.6), p=0.006Protein E% at 9 months was a predictor for weight at 10 years: 0.44 (0.12–0.76), p=0.007, and height 0.51(0.13–0.90), p=0.009.Protein intake (g/day – but not g/kg/day) was a predictor for weight, 0.16 (0.37–0.29), p=0.03 [Wrong in table. Should be 0.16 (0.037–0.29)] and height 0.19(0.042–0.34), p=0.003.	B Measurement errors in dietary recording not considered, power calculation not done
Hoppe, 2004 (13) Denmark cross-sectional	90 children (54 boys, 46 girls)	Protein intake (g/kg/day) at 2.5 years	Associations between protein intake, serum insulin-like growth factor I (sIGF-I) concentrations, and height in in 2.5-year-old healthy children.	The 10th, 50th, and 90th percentiles of protein intake were 2.4, 2.9, and 4.0 g/kg/day, respectively; 63% was animal protein. In multiple linear regressions with adjustment for sex and weight, height (cm) was positively associated with intakes of animal protein (g/day) [0.10±0.038 (b±SE); p=0.01] and milk (0.0047±0.002; p=0.007), but not with those of vegetable protein or meat. The sIGF-I concentration was significantly associated with intakes of animal protein (1.4±0.53; p=0.01) and milk (0.049±0.024; p= 0.045), but not with those of vegetable protein or meat. sIGF-I concentrations were positively associated with height (0.019±0.008; p = 0.02). Milk intake was positively associated with sIGF-I concentrations and height. An increase in milk intake from 200 to 600 mL/day corresponded to a 30% increase in circulating IGF-I. This suggests that milk compounds have a stimulating effect on sIGF-I concentrations and, thereby, on growth.	B Measurement errors in dietary recording not considered, power calculation not done
Koletzko, 2009 (29) European Multicenter study RCT	Children in five countries (Belgium, Germany, Italy, Poland and Spain), n=934 followed until 24 months; 636 in the lower (n=313) and higher (n=323) protein formula groups and 298 in the breastfed group.	Infant formula and follow-on formulas with a lower (1.77 and 2.2 g protein/100 kcal, resp) or higher (2.9 and 4.4 g protein/100 kcal, resp) content of cow milk protein. For comparison, ‘exclusively breastfed’ also followed (<10% of feedings or <3 bottles of formula/week during first 3 months)	Primary outcome: Length, weight at 24 months, expressed as length and weight-for-length z scores based on 2006 WHO growth standards. Secondary outcome: weight, length, weight-for-length and BMI at inclusion, at 3, 6, 12 and 24 months	↑protein →↑weight A higher protein content of infant formula was associated with higher weight in the first 2 year of life but had no effect on length. At 24 months, adj. z score for weight-for-length in the lower protein formula group was 0.20 (95% CI: 0.06, 0.34; p=0.005) lower than in the higher protein group and did not differ from that of the breastfed group. In general, differences were greatest at 12 months for weight, weight-for-length and BMI. Compared with breastfed, those fed high-protein formula had sig higher z scores for weight, length, weight-for-length and BMI at 24 months. Intervention effect did not differ between countries.	A parents lost to follow-up had lower education, mothers more likely to be smokers, BUT no difference between study arms. No differences for those excluded due to non-compliance Biomarkers not used
Kourlaba, 2008 (42), GENESIS Cross-sectional	Uncertain about final number analysed; between 2033 and 2346	Energy and macronutrient intake, including protein, children 1–5 years	Interaction effect between angiotensin-converting enzyme 1 (ACE) 1/D polymorphism and diet on obesity-related phenotypes. DNA samples from 2102 children (1–5 years) were genotyped for the ACE I/D polymorphism; 3 genotypes (ll, lD, DD)	↑protein →↑BMI Significant interactions found between the ACE I/D polymorphism and protein intake on BMI and being overweight (p<0·05 for interaction). Stratified analyses revealed that total energy is correlated with WC and protein intake is associated with BMI and being overweight only among carriers of the D-allele (i.e. DD or ID genotypes). Protein intake was found to be positively associated with the likelihood of being overweight and with BMI (marginally) among the DD homozygotes. Protein intake was higher among ‘at risk of being overweight’ or ‘overweight’ compared with their normal-weight counterparts.	B No power calculation (They′ve used Bonferroni correction, but it is not clear how)
Larnkjær, 2009 (30) Denmark Randomized trial	Healthy infants (n=83) randomized to either whole milk (WM) or infant formula (IF)	Infants randomized to either WM or IF (and either a daily fish oil supplement or no supplement. (2×2 design)	Weight and length at 9 and 12 months and increase in weight and length. IGF-l (insulin-like growth factor l)	WM or IF no effect on change in weight and length. Intake of WM sign increased the protein energy percentage (PE%; p<0.001) and SUN (p=0.01). PE% was 14.2 in WM and 11.4 in IF at 12 months. But no effect of the milk intervention on change in weight or length. Intake of fish oil had no effect on the outcomes.	B Dropout infants did not differ in birth or breast-feeding characteristics from those who finished the study, but they were 1.6 cm shorter at 9 months (95% CI 3.01, 0.21, p=0.039). This could be problematic as it is a study about growth.
Maillard, 2000 (43) France Cross-sectional	501; 280 boys, 221 girls (aged 5±11 years)	Dietary intake at 5–11 years Energy, protein and other nutrients RQ: associations between several adiposity indices and the nutrient intake The associations were looked for according to current dietary recommendations, and according to reported energy intake to basal metabolic rate ratios (EI=BMR) and gender.	Height and weight, four skinfolds (biceps, triceps, subscapular, suprailiac), waist and hip girths, were measured. Sum of skinfolds (SSF), body mass index (BMI), and relative weight (RW) were calculated. Energy intake (EI), percentage of energy intake ascribed to carbohydrates (%EIC), complex carbohydrates (%EICC), fats (%EIF), saturated fats (%EISF) and proteins (%EIP)	In multiple linear regressions analyses performed with hierarchical mixed models, adiposity indices were significantly and inversely associated in girls with %EIC (all p-values < 0.02), and positively with %EIF (all p-values <0.05, waist girth and BMI excepted). Similar but non-significant trends were observed in boys. The relationships were not linear, and thresholds close to current dietary recommendations were highlighted. When%EIF was low, a lower percentage of energy intake ascribed to%EISF was associated with thinness. These associations remained after the exclusion of children who had an EI=BMR _1.50. For both fat and carbohydrate, a substantial percentage of toddlers and preschoolers had usual intakes outside the acceptable macronutrient distribution range, whereas protein was less than this range. ‘At risk of overweight’ and ‘overweight’ children consumed more total energy, protein, and fat compared with their normal-weight counterparts, whereas no differences were found for micronutrient intakes. The estimated prevalence of inadequacy was found to be between 10 and 25% for niacin, vitamin E, and folate. Usual intakes exceeding the Tolerable Upper Intake Levels were recorded for zinc and copper.	B Confounders not taken adequately into consideration
Manios, 2008 (44) Greece Cross-sectional (GENESIS cohort)	2374, age 1 to 5 years	Describe nutrient intake: (a) usual energy and macronutrient intake in the total population as well as by children's weight status, and (b) inadequate or excessive nutrient intakes compared with children's requirements.	Anthropometrical indexes (i.e. body weight, recumbent length, and stature) obtained and BMI was calculated The Nutstat module of EpiInfo was used to determine children's age- and sex-specific percentiles for weight, length, and body mass index. The weight-for-length percentiles were used to classify children up to 24 months old as ‘overweight’ (≥95th percentile), whereas children older than 24 months were classified as ‘at risk of overweight’ (≥85th and <95th percentile) and ‘overweight’ (≥95th percentile) using the body mass index-for-age percentiles.	For both fat and carbohydrate, a substantial percentage of toddlers and preschoolers had usual intakes outside the acceptable macronutrient distribution range, whereas protein was less than this range. ‘At risk of overweight’ and ‘overweight’ children consumed more total energy, protein, and fat compared with their normal-weight counterparts, whereas no differences were found for micronutrient intakes. The estimated prevalence of inadequacy was found to be between 10 and 25% for niacin, vitamin E, and folate. Usual intakes exceeding the Tolerable Upper Intake Levels were recorded for zinc and copper.	B Participation rate not clear, study power not reportedNOTE: no difference in protein E% between the groups (normal weight 17.1±1.6, at risk OW 17.1±1.5, OW 17.1±1.5)
Morgan, 2004 (37) UK Prospective cohort	144	1. Total red and white meat intake (g) from 4 to 12 months as a continuous variable, i.e. total meat intake over 21 days between 4 and 12 months.2. Total red and white meat intake (g) from 4 to 16 months as a continuous variable, i.e. total meat intake over 28 days between 4 and 16 months.3. Total red and white meat intake (g) from 4 to 24 months as a continuous variable, i.e. total meat intake over 42 days between 4 and 24 months.	Body weight, length, and head circumference at the ages of 4, 8, 12, 16, 20 and 24 months	Meat intake from 4 to 12 months was positively and significantly related to weight gain (p<0.05); further analysis suggested this association might be mediated via protein intake but was independent of energy, zinc or iron intake. There was no interaction between meat intake and breastfeeding on growth. These findings remained after adjustment for potential confounding factors.	B Loss to follow-up not reportedNo power calculations
Öhlund, 2010 (4) Sweden Prospective cohort study	127 healthy children (63 girls and64 boys) at 4 years of age followed prospectively from 6 to 18 months of age	Current and previous dietary intake	Weight, height BMI, Mid-upper arm circumference, subcutaneous fat at 4 years of age	Fourteen percent of the girls and 13% of the boys were overweight (age-adjusted BMIX25) and 2% of the girls and 3% of the boys were obese (age-adjusted BMIX30). Thirty-four percent and 9% of the fathers and 19 and 7% of the mothers were overweight and obese, respectively. BMI at 6–18 months was a strong predictor of BMI at 4 years. Intake of protein in particular, and also of total energy and carbohydrates at 17/18 months and at 4 years, was positively associated with BMI at 4 years. Although BMI at 6–18 months was the strongest predictor of BMI at 4 years, in the final multivariate models of the child's BMI, protein intake at 17–18 months and at 4 years, energy intake at 4 years and the father's—but not the mother's—BMI were also independent contributing factors	B Loss of follow-up more than 20% No power calculation
Räihä, 2002 (5) Finland CT	113 term infants, breast-fed and formula-fed.	Parents were instructed to exclusively breastfeed or feed the assigned formula up to 120 days of age (3 isocaloric formulas differing by their protein source and content were studied and compared with breast milk) Calculated energy and protein intakes	increment in anthropometrics parameters from 30 up to 120 days of age (unit/month). Body weight and length were obtained at birth, at 30, 60, 90, and 120 days. Blood was collected for biochemical measurements at 30, 60, and 120 days.	No differences were found between the four feeding groups for weight- and length-gains or for body mass indices (BMI). No differences in energy intakes between the formula fed groups could be found, whereas protein intakes were less in infants fed the 1.8 g/100 kcal formulas. Plasma urea levels of the infants fed the 1.8 g/100 kcal formulas were closer to those found in the breast-fed infants.	B Randomization method not stated adequately Differences between drop-outs and participants nor reported
Sandström, 2008 (31) Sweden CT/Partly RCT	80 (HealthyGA: 36–42 weeksBWT: 2500–5000 g)	Standard vs. two formulas varying in G Lycomacropeptide (GMP) and α-lactalbumin i.e. 3 formulas w. bovine whey fractions rich in α-lactalbumin w. varying GMP vs. breast feeding (as control) All formulas: 1,96 g prot/ 100 kcal.	-Growth -General health -Plasma leptin, insulin, urea nitrogen, amino acids	Formula intake was similar in different groups. Weight gain in the alpha-lactalbumin-enriched formula groups was similar to that of the breastfed infants. The standard formula group gained significantly more weight than did the breastfed infants. All formula-fed infants had significantly higher plasma concentrations of most essential amino acids at 4 and 6 months than did the breastfed infants, and serum urea nitrogen was also higher in the formula-fed infants. Insulin and leptin concentrations did not differ between groups.	B No power calculation reported, compliance unclear, energy intake unclear, results not analysed blind, unclear about between measurements errors
Scaglioni, 2000 (38) Italy Prospective cohort	147	Nutrients/ early macronutr. Intake, Parental factors	Anthropometry at 1, 5 years	The prevalence of overweight at the age of 5 years was strongly associated with parental overweight (p<0.0001). Five-year old overweight children had a higher percentage intake of proteins at the age of 1 year than non overweight children (22 vs. 20%, p=0.024). Multiple logistic analysis confirmed that protein intake at 1 year-of-age was associated with overweight at 5 years (p=0.05). In children born from overweight mothers, prevalence of overweight at the age of 5 years tended to be higher in bottle-fed than in breast-fed ones (62.5 vs. 23.3%, p=0.08). Conclusion: Parental overweight is a major risk factor for childhood overweight in the first years of life, but an early high protein intake may also influence the development of adiposity.	B Measurement errors in dietary reporting not considered Energy intake little bit high No power calculation
Skinner 2004, (39) Prospective cohort	70	Energy and macronutrient intakes at each study point	BMI, age of adiposity rebound was determined	Children's BMI at 8 years was negatively predicted by age of adiposity rebound and positively predicted by their BMI at 2 years. Mean protein and fat intakes recorded between 2 and 8 years were positive predictors of BMI at 8 years; mean carbohydrate intake over the same time period was negatively related to BMI at 8 years. R2 values indicated that these three-variable models predicted 41–43% of the variability in BMI among children. BMI of 23% of the children exceeded the 85th CDC percentile.	B Baseline not clearly indentified, easurement errors in dietary recording not considered, power calculation not done
Van Vught, 2010 (40) Denmark Prospective cohort	223	Protein intake, especially the amino acids: Lysine (LYS) Arginine (ARG)	Growth & body composition Fat mass index (FMI)	No association between protein intake and linear growth. However, amino acids could be important. High ARG intake, but not LYS, was associated with linear growth (β = 1.09 (se 0.54), p=0.05) among girls. Furthermore, in girls, change in FMI had a stronger inverse association with high ARG intake, if it was combined with high LYS intake, instead of low LYS intake (p=0.03). No associations were found in boys. In prepubertal girls, linear growth may be influenced by habitual ARG intake and body fat gain may be relatively prevented over time by the intake of the amino acids ARG and LYS.	B Energy adjustment not done No power calculation
Van Vught, 2009 (41) Denmark Prospective cohort	384 of originally 771	Diet Protein, amino acids: ARG, LYS	Skinfold thickness was measured at ages 8–10 years and 14–16 year. BMI and Body fat% was estimated from skinfold measurements	Among lean girls inverse associations were found between protein as well as arginine and lysine intake and change in fat mass index (β = −1.12 + /−0.56, p=0.03, β = −1.10 + /−0.53, p=0.04, β = −1.13 + /−0.51, p=0.03 respectively). Furthermore among girls with a body mass index in the 5th quintile, protein intake was associated with DeltaFFMI (p=0.04), and more specific when LYS intake was high, ARG intake was associated with DeltaFFMI (p=0.04). No associations were found in boys.	B Measurement errors in dietary reporting not consideredOnly 49.8% of original sample took part in baseline (57% of those still living in area took part in follow-up)