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Variation in Fat, Lactose, and Protein Composition in Breast Milk over 24 Hours : Associations with Infant
Feeding Patterns
Sadaf Khan, Anna R. Hepworth, Danielle K. Prime, Ching T. Lai, Naomi J. Trengove and Peter E. Hartmann
J Hum Lact 2013 29: 81 originally published online 13 July 2012
DOI: 10.1177/0890334412448841

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448841
841Khan et al.Journal of Human Lactation
JHLXXX10.1177/0890334412448

Original Research
Journal of Human Lactation

Variation in Fat, Lactose, and Protein 29(1) 81­–89

© The Author(s) 2013
Reprints and permission: http://www.
Composition in Breast Milk over 24 sagepub.com/journalsPermissions.nav
DOI: 10.1177/0890334412448841

Hours:  Associations with Infant Feeding

http://jhl.sagepub.com

Patterns

Sadaf Khan, MSc1, Anna R. Hepworth, BSc (Hons), Dip Ed1,

Danielle K. Prime, PhD1, Ching T. Lai, PhD1, Naomi J.Trengove, PhD1,
and Peter E. Hartmann, PhD1

Abstract
Background: Data regarding the association between breast milk composition and infant feeding patterns (frequency and
amount of breast milk taken) would help in understanding the regulation of food intake in breastfed infants.
Objective: This study examined the relationship between breast milk macronutrient concentration and patterns of milk
intake in breastfeeding infants over a 24-hour breastfeeding period.
Methods: Mothers of healthy term infants (n = 15) collected pre- and postfeed breast milk samples from each feed at each
breast over a 24-hour period. Breast milk samples were analyzed for fat, lactose, total protein, casein, and whey protein
content. The energy content for each feed was calculated.
Results: Breastfeeding patterns and milk composition varied greatly between individuals. The fat content of milk significantly
differed over 24 hours (P = .01), whereas the concentration of lactose and protein content remained the same. The mean
24-hour total protein, whey, and casein intake was inversely (P < .01), whereas lactose concentration was positively (P = .03)
related to the number of breast feeds per day. No relationship was seen either between fat or energy content and feeding
patterns. The mean (SD) concentration of fat, lactose, and total protein over the 24-hour period was 43 (12) g/L, 68 (7) g/L,
and 13 (2) g/L, respectively.
Conclusion: The association between milk protein intake and the breastfeeding frequency suggests that the protein intake
may play a role in infant appetite control.

Keywords
breastfeeding, energy, fat, feeding patterns, lactose, milk intake, protein

Well Established Background

It has been well established that breastfed infants self-regulate In exclusively breastfeeding mother-infant pairs, wide varia-
their milk intake and feed according to their appetite. However, tions have been reported in breastfeeding frequency, volume
data regarding the changes between breast milk composition of milk removed at each breastfeed, and distribution of milk
and infant’s level of demand are limited. intake both during the day and night.1-3 Kent et al1 reported

Newly Expressed Date submitted: September 9, 2011; Date accepted: April 17, 2012.

We investigated the association between breast milk composi- 1

tion and infant feeding patterns over a 24-hour breastfeeding
period.The results showed a negative relationship between milk
proteins and frequency of breast milk intake, which suggests
that protein intake may play a role in regulation of food intake
in infants.
The University of Western Australia, Crawley, Western Australia, Australia
Corresponding Author:
Sadaf Khan, School of Chemistry and Biochemistry, The University of
Western Australia, M310, 35 Stirling Hwy, Crawley, Western Australia,
Australia
Email: khans01@student.uwa.edu.au

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82 Journal of Human Lactation 29(1)
a breastfeeding frequency of 11 ± 3 times in a 24-hour
period with a breast milk intake of 76 ± 13 g per feed. In
addition, there was also considerable variation in daily milk
intake of breastfed babies with a reported range of 500 to
1300 mL/d.1,3,4 These variations in the breastfeeding patterns
suggest the existence of a self-regulatory mechanism that
determines milk intake from feed to feed in breastfed
infants, allowing them to feed according to appetite.
Previous studies have shown that breastfed infants con-
sume on average 67% of the available milk at each breast-
feed, indicating that milk intake is not restricted by maternal
milk supply and is determined according to infant demand.1,5
In addition, a positive relationship was identified between
volume of milk consumed at a feeding and preprandial
interval in breastfed infants, further suggesting that breast-
fed infants determine their feeding size, intervals, and fre-
quency to regulate their intake.6,7
Large numbers of potential factors may influence the milk
intake in infants. Milk consumption by breastfed infants can be
affected either by maternal (eg, variation in milk composition
and production) or infant-related factors (eg, body weight,
growth-related metabolic requirements, stomach storage capac-
ities, and gastric emptying rates) or a combination of both.
Data regarding the association between milk composition
and the infant’s level of demand are limited. There are several
studies concerning the regulation of milk intake in infants,
but most of them are in relation to maternal milk supply.4,5
Some studies have investigated the influence of fat content,
because it is the major constituent of energy and the most
variable component of breast milk, yet these studies failed to
establish the change in fat during the course of the feed as a
signal for the baby’s appetite control mechanism.8,9
Along with hormonal factors, gastric emptying is consid-
ered a key regulator of appetite since rapid emptying can
lead to a much earlier sensation of hunger.10 Different
behavior and gastric emptying rate of each type of milk pro-
tein (casein curd and soluble whey proteins) during diges-
tion11 suggest that the protein composition may either play a
role in the different feeding patterns seen among breastfed
infants or have a link to infant cues for hunger. Therefore,
there is a need to investigate how infant feeding patterns are
influenced by the 3 energy-yielding macronutrients (fat,
protein, and lactose) with particular emphasis on the protein
composition (casein and whey proteins). This study aimed
to examine the association between the variation of breast
milk macronutrient concentration over a 24-hour breast-
feeding period and the patterns of milk intake in infants.
Materials and Methods
Participants
Breast milk samples were obtained from 15 mothers of
healthy, term infants recruited through the Western Australian
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branch of the Australian Breastfeeding Association and the
Child and Adolescent Community Health Nurses in the
Oceanic region. Infants were all exclusively breastfed on
demand, and had a mean (SD) age of 3.2 (1.6) months
(range, 1.0-6.0 months). All participating mothers measured
their infant’s 24-hour milk intake and provided written
informed consent to participate in the study, which was
approved by the Human Research Ethics Committee of The
University of Western Australia.
The number of participants was selected on the basis of
two considerations, which were to include sufficient partici-
pants to cover the variation in the population and to have
data for at least 150 feeds. For these, 15 participants was
considered a minimum requirement based on an assumption
that on average, each participant would provide the samples
for 11 ± 3 feeds,1 and this would provide the samples or data
for approximately 165 feeds.
Milk Sampling
Milk samples (1-2 mL) were collected before (pre) and
immediately after (post) each feed, from each breast, over
a 24-hour period by either manual breast pump or hand
expression into 5-mL polypropylene vials (Disposable
Products Pty Ltd, Adelaide, Australia). Samples were ini-
tially stored in the mother’s home freezer for a maximum
of 24 hours and then transported on ice to the laboratory
where they were stored at –20°C until analyzed.
Biochemical Analysis
Milk fat. The fat content of pre- and postfeed milk samples
was analyzed by creamatocrit method12 using the Creamato-
crit Plus device (Medela, Inc, McHenry, Illinois). This
method has been demonstrated to have a strong correlation
with results obtained by the spectroscopic esterified fatty
acid (EFA) assay.13,14
Milk lactose. The lactose content of pre- and postfeed milk
samples was determined by an enzymatic spectrophotomet-
ric method.14,15 The recovery of a known amount of lactose
added to milk samples was 101% (standard error [SE] 0.5%,
n = 12). The detection limit (3*SD low concentration stan-
dard) of this assay was 0.019 g/L (n = 20), and the interassay
coefficient of variation (CV) was 4.2% (n = 60).
Milk protein. Previous analysis of changes in protein
concentration in milk samples has shown no significant
difference between pre- and postfeed samples16; there-
fore, milk protein analysis was carried out on pre- and
postfeed milk samples that were pooled. Samples were
defatted and then subjected to protein separation to obtain
casein and whey proteins according to the method
described by Kunz and Lonnerdal.17,18 The protein con-
centration of skim (defatted milk), whey, and casein frac-
tions of the samples was determined by the Bradford protein
Khan et al. 83
assay using a commercial protein reagent (Bio-Rad Labo-
ratories, Richmond, California). Human milk protein
standards were prepared by determining the concentration
of an aliquot of mature breast milk as described by Atwood
and Hartmann.19 The protein assays were carried out by
the procedure described by Mitoulas et al.14 Skim milk
and whey samples were diluted 1 in 30, and for casein
samples, a 1 in 10 dilution was used. The recovery of a
known amount of the protein added to the milk samples
was 99.8% (SE 1.4%, n = 12). The detection limit of the
assay was 0.045 g/L (n = 30), and the interassay CV was
6.4% (n = 50).
Milk energy. The energy content for each breastfeed was
calculated using the conversion factors of 9.0, 4.0, and 4.0
kcal/g for fat, protein, and lactose, respectively.20
Measurement of 24-Hour Infant Milk Intake
Infant milk intake from each breast was determined by
the test-weighing procedure.21 Mothers test weighed their
infants before and after each feed, from each breast, using
electronic baby weigh scales (Medela AG, Baar,
Switzerland; resolution 2 g, accuracy ±0.034%). Infant
milk intake was calculated by subtracting the initial
weight of the baby from the final weight of the baby. Test
weighing was done at each mother’s home for a period of
24 to 28 hours. The measurements were later corrected to
24 hours with no correction made for infant insensible
water loss; therefore, milk intake may be underestimated
by a mean (SD) of 10% (12%).21 Degree of breast fullness
and amount of milk available in the breast were calcu-
lated from these data as described previously.1 Briefly,
because there is a relationship between the fat content of
the milk and the degree of fullness of the breast,22 mea-
suring the fat content of the samples allows the calcula-
tion of the degree of fullness of the breast before and after
each breastfeeding. Degree of fullness was calculated as
1 – degree of emptying using the equation described by
Daly et al.22 Minimal and maximal fat content over 24
hours corresponds to degree of fullness of 1 and 0,
respectively.
Since mothers were breastfeeding on demand, for this study,
the 24-hour period was divided into 4 intervals of 6 hours and
defined as morning (4:01 AM to 10:00 AM), day (10:01 AM to
4:00 PM), evening (4:01 PM to 10:00 PM), and night (10:01
PM to 4:00 AM).1
Determination of 24-Hour Nutrient and
Energy Intake
The protein, lactose, and energy intakes of each infant were
calculated from 24-hour milk intake and from milk composi-
tion data. In addition, the 24-hour fat intake was determined
as described by Mitoulas et al.14 The concentration of fat for
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each feed was obtained by averaging the pre- and postfeed
milk concentrations. The fat intake at each feed was then
determined from the volume of the feed. The sum of the
intake of fat for all feeds and the total volume consumed over
the study period (24-48 hours) was then used to determine an
average concentration. This average concentration and the
corrected 24-hour volume21 were then used to determine the
infant total fat intake in the 24-hour period.
Statistical Analysis
Statistical analysis was performed using the R program, ver-
sion 2.7.2.23 The package nlme24 was used for linear mixed
effect models.
The differences between macronutrient concentrations
and intake over the 24-hour period were tested using linear
mixed models with the inclusion of time of the day as a fac-
tor. Pre- and postfeed milk samples were also compared
using linear mixed models.
Univariate relationships between milk intake variables
(such as frequency of breastfeeding, breastfeed volume,
duration of feed, interval between feeds, 24-hour milk
intake), milk composition, and intake were tested with lin-
ear mixed effects model.
In all analyses, different intercepts of each individual
and breast within mother were used as a random effect in
the model. Age of infant was tested as univariate predictor,
and where a significant relationship was seen, it was
included as a covariate in the model for that nutrient (the
only factor for which this was significant was the whey
protein concentration [P = .048]).
Student paired t test was used to compare the milk intake
variables. Data were normally distributed and are presented
as mean (SD), unless otherwise stated. All values are reported
as mean (SD), unless otherwise stated. P values less than .05
were considered statistically significant.
Results
Breastfeeding Characteristics
The milk intake patterns of all 15 individual mother-infant
pairs over a 24-hour period are presented in Figure 1. The
mean (SD) 24-hour milk intake of the infants was 802
(161) g (range, 598-1113 g) and was similar between the
left (400 [128] g) and right (402 [104] g) breasts. The num-
ber of breastfeeds during the day ranged from 11 to 19 (15
[3] feeds per 24 hours), and the mean (SD) milk intake of
the infants at a breastfeed was not significantly different
from the left (64 [16] g) and right (62 [18] g) breasts.
Furthermore, there was no difference between left and
right breasts in the time between feeds (left breast, 1.56
[1.8] hours; right breast, 1.60 [1.9] hours) during the
24-hour period.
84 Journal of Human Lactation 29(1)

Figure 1. Patterns of Breastfeeds over 24 Hours in Individual Mother-Infant Pairs (n = 15).

Each box of the lattice plot represents a single mother-infant pair, and symbols represent the feed from left (*) and right (o) breasts.

Milk Fat
The average milk fat content was 43.2 (11.8) g/L, ranging from
28 to 57 g/L. Significant differences were observed in the fat
concentration of pre- and postfeed milk samples (32 [12] g/L,
56 [17] g/L; P < .001), respectively (Figure 2). Overall, there
were higher levels of fat in the postfeed milk, with an average
difference of 24 g/L between post- and premilk fat content.
There were significantly different baseline fat contents between
women as well as between breasts from each woman (on aver-
age). Analysis of fat content at 4 time periods within 24 hours
(morning, day, evening, and night) showed that fat content was
significantly related to time of day (P = .01). Compared with
the morning samples, fat content was higher during the day
(P = .01) and lower at night (P = .02), with no difference
between morning and evening fat concentration.
The average fat content was not associated with the inter-
vals between feeds (P = .15), the number of breastfeeds dur-
ing the day (P = .78), volume consumed during the feed
(P = .11), the 24-hour milk intake from each breast (P =
.47), or feed duration (P = .40). The average 24-hour fat
intake of the infant was 34.8 (8.4) g (range, 16-48 g) and
was not related to the duration of the feed (P = .67) or the
frequency of breastfeeds (P = .13).
Milk Lactose
The average concentration of lactose in milk was 68 (6.8) g/L
and ranged from 59 to 76 g/L. No significant difference was
observed in lactose concentration in pre- and postfeed milk
samples (P = .33) (Figure 2) and also between left (pre, 69.0
[7.5] g/L; post, 68.0 [8.8] g/L) and right (pre, 68.0 [7.7] g/L;
post, 67.6 [7.4] g/L) breasts. Analysis of lactose concentration
over the 4 time periods within 24 hours (morning, day, eve-
ning, and night) showed that lactose concentration was similar
at all time points of the day (P = .62). Furthermore, the aver-
age lactose concentration was not associated with the intervals
between feeds (P = .41), the duration of the feed (P = .21), the

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Khan et al. 85

Figure 2. Distribution of Fat and Lactose Concentration in Pre- and Postfeed Milk Collected during Breastfeeding over 24 hours (n =
226 samples from 15 mothers)

Values are shown by box plots illustrating median (indicated by the bold line), quartiles (box), range (error bars), and outliers (o).
a
Indicates a significant difference (P < .05).

volume consumed during the feed (P = .57), or the amount of
available milk in the breast (P = .21). However, there was a
positive relationship between the average lactose concentra-
tion of the milk and 24-hour milk production from that breast
(P = .02). In addition, a significant positive relationship was
found between lactose concentration and the number of
breastfeedings during the day (P = .03), with higher lactose
concentration associated with more feeds per day.
The average 24-hour lactose intake of the infants was
56.5 (14.7) g (range, 29.4-78.1 g) and was not related to the
duration (P = .79) or frequency of the feeds (P = .97).
Milk Protein
The average protein concentrations for all mothers were
13.4 (2.2) g/L (range, 10-17 g/L), 7.6 (1.5) g/L (range, 5-9
g/L), and 3.4 (1.0) g/L (range, 2-5 g/L) for skim milk, whey
protein, and casein, respectively.
No significant differences were observed in protein con-
centration of skim milk, whey protein, and casein fraction
over the 24-hour period.16 Furthermore, the interval between
feeds, duration of the feed, volume consumed during the
feed, breastfeed frequency, and amount of available milk in
the breast were not associated with average protein concen-
tration of skim, whey protein, and casein fraction.
The average 24-hour total protein intake of the infants
was 10.9 (2.62) g (range, 7.3-17.6 g) and was independent
of the duration and interval between feeds. A significant
negative relationship was found between the 24-hour total
protein intake and frequency of breastfeeds (P = .01). Thus,
a higher 24-hour protein intake was significantly associated
with fewer feeds per day.
The mean 24-hour whey protein and casein intake was 4.5
(2.1) g (range, 2.3-9.7 g) and 2.1 (1.1) g (range, 0.9-5.2 g),
respectively. Both the 24-hour whey protein and the casein
intake were negatively associated with the frequency of the
breastfeeds (P < .01).
Milk Energy
The mean energy content and the amount of energy deliv-
ered to the infant during the 24-hour period were 714 (117)
kcal/L (range, 540-842 kcal/L) and 578 (135) kcal/24 hours
(range, 292-776 kcal/24 hours), respectively.
Energy content was significantly related to time of day
(P = .01). Compared with the morning, the energy content
was higher during the day (P < .01) and lower at night (P = .03).
In addition, a significant negative relationship was found
between the energy content of the milk and degree of breast
fullness (P < .01).22 The energy content was not associated
with intervals between feeds (P = .25), duration of the feed
(P = .50), volume consumed during the feed (P = .18), num-
ber of breastfeeds during the day (P = .83), or the 24-hour
milk intake from each breast (P = .19). Similarly, there was
no significant relationship between the total energy delivered
to the infant and 24-hour milk intake measures.

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86 Journal of Human Lactation 29(1)

Table 1. Milk Composition and Breastfeeding Pattern over a 24-Hour Period

Time of the Day

  Morning Day Evening Night

ab a b
Feed volume, g 75 (34) 59 (29) 53 (26) 75 (36)
Feed duration, min 12.0 (4.9) 10.7 (4.17) 12.2 (4.6) 12.3 (5.0)
Degree of breast fullness
 Prefeed 0.63 (0.23)a 0.55 (0.24)a 0.59 (0.22) 0.72 (0.25)
 Postfeed 0.23 (0.17)ab 0.15 (0.14)a 0.19 (0.16) 0.31 (0.19)b
Fat, g/L
  Average fat 40.4 (11.1)ab 47.8 (12.3)a 43.2 (10.1) 37.9 (10.6)b
 Prefeed 29.3 (10.9)a 35.0 (12.9)a 31.6 (10.4) 28.1 (12.2)
 Postfeed 52.9 (16.3)ab 61.8 (18.8)a 56.3 (15.7) 48.9 (14.4)b
Lactose, g/L
  Average lactose 67.7 (7.6) 67.9 (6.3) 68.5 (6.8) 68.5 (6.9)
 Prefeed 67.9 (8.0) 68.1 (7.0) 69.0 (7.8) 69.0 (8.0)
 Postfeed 67.6 (8.5) 67.6 (7.9) 68.0 (7.7) 68.0 (8.4)
Total protein, g/L 13.0 (2.1) 13.3 (2.1) 13.7 (2.4) 13.4 (1.9)
Whey, g/L 7.5 (1.6) 7.6 (1.5) 7.6 (1.6) 7.6 (1.2)
Casein, g/L 3.3 (1.0) 3.4 (1.0) 3.5 (1.0) 3.6 (1.0)
Energy, kcal/L 687 (116)ab 755 (118)a 716 (104) 669 (110)b
Data presented as mean (SD). Data for left and right breasts for all samples within the time point of the 15 mothers are combined. The superscript letters ab
represent significant difference, such that time point of the day containing the same symbol was significantly different from each other (P < .05).

Discussion Our results showed no association between the changes

This study has examined the association between breastfeed-
ing patterns in exclusively breastfed infants and the macro-
nutrient content (particularly the protein composition) of
their mother’s milk. The 24-hour breastfeeding patterns of
the participants in this study were within the normal range,1
and the mean 24-hour milk intake of these exclusively
breastfed infants was also consistent with the previous stud-
ies for breastfed infants between 1 and 6 months of age.1,4
However, there was considerable variation among the
mother-infant pairs in the frequency of breastfeeding, breast-
feed duration, interval between feeds, and infant milk intake
(Figure 1).
The present study is in agreement with previous studies
showing marked variation in the milk fat content during the
feed and over the course of a 24-hour period (Table 1).14,25-27
Daly et al22 established the relationship between fat content
and extent of breast fullness and showed that approximately
70% of the variation in fat content of breast milk was due to
the changes in the volume of milk available in the breast
before and after each breastfeed.28 Therefore, the 2-fold
increase observed in the fat content (Figure 2) during the
feed was related to the degree of breast emptying (ie, fat con-
tent increased along with the breast empting).22 Similarly,
higher fat content during the day compared with morning
and night was due to a higher degree of milk removal during
the day and a higher degree of fullness during morning and
night (Table 1), which is consistent with previous reports.1,29
in fat content and the milk intake in breastfed infants, which
supports previous research.8,9 Although fat is the most vari-
able macronutrient of breast milk, these results suggests that
changes in fat concentration during the feed and time of the
day are more likely related to the physiochemical adsor-
bance effect (ie, the removal of adsorbed fat globules due to
the change in morphology of lactocytes upon milk ejec-
tion)19,30 rather than to any physiological signals related to
infant appetite cues. Furthermore, fat intake was indepen-
dent of the frequency of breastfeeding during the day, indi-
cating that regardless of the infant patterns of milk intake,
the daily fat consumption is similar between frequent and
infrequent feeders.31 Since fat provides approximately half
of the infant energy intake, infants get the same amount of
energy from their mother’s milk throughout the day regard-
less of their feeding behavior. In addition, the similar pat-
terns of variation observed in milk energy content can be
attributed to the changes in fat content.
Dietary proteins have been shown to suppress the short-
term food intake and have a higher satiety effect than
either carbohydrates or fats in both animals and humans.32
Furthermore, these effects of dietary proteins are also
dependent on the protein source33 and amount consumed
as it has been shown that high-protein diets are more sati-
ating than low-protein diets.34 The milk proteins whey and
casein are classified as “fast” and “slow” proteins, respec-
tively, based on their different effect on food intake in
humans.35 The digestion and absorption of whey protein

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Khan et al. 87
Table 2. Macronutrient Concentration and 24-Hour Infant Nutrient Intake from Left and Right Breasts
Macronutrient Concentration, g/L
Breast Left
Fat 43.1 (12.6)
Total protein 13.4 (2.1)
Whey 7.7 (1.5)
Casein 3.4 (1.0)
Lactose 68.5 (7.0)
Energy, kcal 715 (121)
n = 15. Results are mean (SD). No statistical differences were observed.
amino acids are faster than that of casein.35 Whey con-
sumption also leads to a higher plasma concentration of
satiety hormones such as CCK, GLP-1, and peptide YY.36
In addition, the difference between the effects of whey
proteins and casein on food intake may also depend on
their different behavior during digestion. Whey proteins
are rapidly emptied into the small intestine as intact pro-
teins, whereas casein proteins are slowly emptied from the
stomach, mainly in the form of degraded peptides. This
difference in their behavior is due to the clotting and/or
precipitation of the casein (unlike the soluble whey pro-
teins) in the acidic environment of the stomach, resulting
in higher exposure of casein to gastric peptic hydrolysis.37
However, despite the slow and fast behavior of casein and
whey proteins during digestion, Luhovyy et al38 suggested
that the combined action of whey and casein contributes to
the satiety effect and the delay of the return of hunger, with
whey providing early signals and casein giving overlap-
ping but late signals. In the present study, total protein
intake and intake of both whey protein and casein in 24
hours were found to have a significant inverse relationship
with breastfeeding frequency. Overall, this is in agreement
with a previous study in rats that showed that the whey
protein diet is more effective in increasing the interval
between meals and reducing the meal frequency compared
with other protein sources (soy and gluten).33 This sug-
gests that milk proteins may have a role in the regulation
of body weight through their satiety signals that can affect
both short- and long-term regulation of food intake.38
Formula-fed infants were shown to have higher levels of
plasma insulin than breastfed infants, which can stimulate
fat deposition and early development of adipocytes.39 It
has been suggested that this change in the infant’s hor-
monal status, which is possibly due to higher protein levels
of milk formulas compared with breast milk, may also
contribute to either higher energy intake or growth veloc-
ity in formula-fed infants.40 The protein hormones such as
leptin, ghrelin, and obestatin, which are associated with
energy intake and expenditure, have also recently been
found in human breast milk.41,42 Although we did not
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Macronutrient Intake, g/24 h
Right Left Right
43.3 (10.9) 17.3 (6.0) 17.5 (4.9)
13.4 (2.3) 5.5 (1.8) 5.4 (1.5)
7.5 (1.5) 2.2 (1.1) 2.3 (1.2)
3.4 (1.0) 1.0 (0.64) 1.0 (0.56)
67.8 (6.6) 28.5 (10.1) 27.9 (8.3)
713 (113) 290 (96) 289 (81)
assess the individual proteins, the presence of these bioac-
tive peptides in breast milk and the association between
protein intake and metabolic regulation further strengthen
the possible role of protein components in the control of
appetite in developing infants. In addition, the protein
intake was highly variable between infants. Therefore, it is
possible that this may contribute to the differences in feed-
ing patterns as well as growth patterns of an infant, but
these are areas that require further investigation.
There were no differences in the lactose concentration
during the feed, between breasts, and throughout the day
(Figure 2 and Table 1), and this is consistent with the
reports of invariant lactose concentrations in milk.14,25
However, a relationship was found between lactose con-
centration and breastfeeding frequency, and this may be
explained by the fact that high-lactose feeds are associated
with rapid gastric emptying.43 In addition, the positive
association between the lactose concentration and number
of breastfeeds per day is consistent with the relationship
between lactose concentration and the 24-hour milk pro-
duction of the mothers. This relationship has also been
reported previously15,44 and might be due to the contribution
of lactose to milk osmolarity.44
The sampling protocol used in the study was similar to
Hartmann et al45; we took into account all levels of varia-
tions in milk components during feeding, between breasts,
and over the 24-hour period and accurately determined the
nutrient intake by the infants with minimal disturbance to
infant feeding behavior. Therefore, the mean 24-hour infant
intake of fat, lactose, and protein determined in this study
(Table 2) agreed with the range of estimated intakes reported
by others at a similar stage of lactation.3,14 Due to the differ-
ent digestibility of casein and whey proteins, it has been
suggested that low casein content in milk during early lacta-
tion may facilitate digestion and could be an advantage for
the immature digestive system of newborn infants.46 The
limitation of this study includes the lack of follow-up at dif-
ferent stages of lactation, but previous studies have shown
that the casein:whey ratio in human breast milk is relatively
stable during established lactation as compared with early
88 Journal of Human Lactation 29(1)
lactation.47 To our knowledge, no studies have compared the
mean 24-hour intake of casein and whey proteins in breast-
fed infants. However, to make dietary recommendations
regarding these proteins requires further studies, and prefer-
ably longitudinal studies with specific functional outcomes
(such as the relation of whey protein and casein to growth
patterns in infants) are required.
Conclusion
In summary, the association between milk proteins (whey
protein and casein) and frequency of breast milk intake
suggests that protein composition may have a role in the
regulation of food intake and necessitates studying both
the short- and long-term effect of protein on infant growth.
Apart from the dietary constituents of a feed, several inter-
acting factors are involved in the regulation of appetite
and energy intake, including gastrointestinal and hor-
monal factors. Therefore, to completely understand the
association between feeding patterns in breastfed infants
and appetite control physiology, it is important to consider
all of these aspects, particularly gastric emptying rates and
the infant’s stomach storage capacities. The complete
understanding of early programming of infant appetite
needs to be explored as infant feeding practices have the
potential to contribute to the development of infant and
child obesity.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The authors disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: This
study was supported by the Higher Education Commission
(Pakistan) and an unrestricted research grant from Medela AG.
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