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ABSTRACT BACKGROUND Human scalp hair is a validated bio-substrate for monitoring various exposures in childhood including contextual stressors, environmental toxins, prescription or

non-prescription drugs. Linear hair growth rates (HGR) are required to accurately interpret hair biomarker concentrations. METHODS We measured HGR in a prospective cohort of preschool

children (_N_ = 266) aged 9–72 months and assessed demographic factors, anthropometrics, and hair protein content (HPC). We examined HGR differences by age, sex, race, height, hair pigment,

and season, and used univariable and multivariable linear regression models to identify HGR-related factors. RESULTS Infants below 1 year (288 ± 61 μm/day) had slower HGR than children aged

2–5 years (_p_ = 0.0073). Dark-haired children (352 ± 52 μm/day) had higher HGR than light-haired children (325 ± 50 μm/day; _p_ = 0.0019). Asian subjects had the highest HGR overall (_p_ = 

0.016). Younger children had higher HPC (_p_ = 0.0014) and their HPC-adjusted HGRs were slower than older children (_p_ = 0.0073). Age, height, hair pigmentation, and HPC were related to HGR

in multivariable regression models. CONCLUSIONS We identified age, height, hair pigment, and hair protein concentration as significant determinants of linear HGRs. These findings help

explain the known hair biomarker differences between children and adults and aid accurate interpretation of hair biomarker results in preschool children. IMPACT * Discovery of hair

biomarkers in the past few decades has transformed scientific disciplines like toxicology, pharmacology, epidemiology, forensics, healthcare, and developmental psychology. * Identifying

determinants of hair growth in children is essential for accurate interpretation of hair biomarker results in pediatric clinical studies. * Childhood hair growth rates define the

time-periods of biomarker incorporation into growing hair, essential for interpreting the biomarkers associated with environmental exposures and the mind-brain-body connectome. * Our study

describes age-, sex-, and height-based distributions of linear hair growth rates and provides determinants of linear hair growth rates in a large population of children. * Age, height, hair

pigmentation, and hair protein content are determinants of hair growth rates and should be accounted for in child hair biomarkers studies. * Our findings on hair protein content and linear

hair growth rates may provide physiological explanations for differences in hair growth rates and biomarkers in preschool children as compared to adults. You have full access to this article

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INTRODUCTION Discovery of hair biomarkers in the past few decades has transformed scientific disciplines like toxicology, pharmacology, epidemiology, forensics, healthcare, and developmental

psychology.1,2,3,4 Moreover, the frequent use of hair in large population cohorts to measure environmental exposures in children has emerged from the discovery of hair biomarkers.5,6,7,8,9

Cortisol in human scalp hair is a validated biomarker for monitoring time-averaged acute/chronic responses to various familial, health, psychosocial, and economic conditions across the

lifespan.10,11,12,13,14 Hair biomarkers capture these ecological exposures and can serve as organic record logs of various exposures.11,15,16,17 To identify the time-periods for these

exposures, it is important to understand the determinants of hair growth and adjust for confounding factors.18,19,20,21 Scalp hair is characterized by growth cycles of active growth

(anagen), transition (catagen), and resting (telogen) phases; hair density, diameter, or linear length; and even according to its texture (straight or curly), shape, pigmentation, or

elasticity.22,23,24,25 These characteristics may reflect nutritional status, mitochondrial metabolism, puberty, pregnancy, or seasonal changes.18,25,26,27,28,29 Furthermore, a few hair

studies demonstrate the diversity in hair quality and character across individual factors, such as race, ethnicity, age, sex, and disease.2,11,30,31,32 Studies of hair growth have been

reported on exceptionally few children and focused on keratinization, medullation, or diameter/cross-sectional growth patterns.22,33,34,35,36 Human scalp hair, derived from the neuroectoderm

and mesoderm,34,37 evolves via prenatal lanugo, postnatal vellus, intermediate medullary, and post-pubertal terminal hair stages.24,25,38 These studies also revealed that: (a) scalp hair

medullation increases diameter with the greatest increases occurring at 1–3 years of age and no sex differences in hair diameter; (b) males have greater hair density (follicles/cm2) and

faster linear hair growth rates at 3–9 years.22,24,36 Linear hair growth rates in adults are correlated with hair diameter, medullation, and loss of pigmentation; microscopically correlated

with the hair follicle size, and interscale distances within the hair shaft.27,39,40 While these are important observations, linear hair growth rates (HGRs) are required to accurately

interpret the time frame during which biomarkers are incorporated into the growing hair shaft, and linear HGRs remain undetermined in children. Current hair biomarker research practices

involve the application of adult HGRs to the interpretation of biomarker levels in children.11,41 Rapid physical growth and development in childhood may alter HGRs; thus, child HGRs may

differ from HGRs reported in adults.27 For example, differing biomarker (cortisol) levels in adults vs. children demonstrated by Neumann et al. and de Kruijff et al. may be due to

age-dependent differences in HGRs.42,43 Race-dependent differences in hair growth linked to hair color, texture, biochemical composition, and hair growth cycles are determined in adults

(specifically in African, Asian, European/Caucasian groups), but remain undetermined in children.44 Child hair biomarker studies have described analogous race-dependent differences linked to

hair qualities.11,38 Furthermore, Barth and Pecoraro et al. reported higher HGRs in male vs. female children (5–15% faster hair growth; 340 vs. 302 μm/day, respectively), but both examined

small sample sizes and only performed descriptive statistical analyses.38,45 On the whole, adult hair studies provide biomarker research with more sophisticated findings (more studies,

larger sample sizes, rigorous methodologies, and statistical analyses) than child hair studies. For example, while the composition of adult hair is known (0.25–0.95% trace elements,1–9%

lipids, 3–5% endogenous water, and 65–95% protein),46,47,48,49,50 the composition of children’s hair largely remains unknown. We designed this study to understand hair growth physiology in

early childhood, specifically investigating anthropometric measures and demographic factors that determine linear HGRs in preschool children. Results from this study will enable better

interpretation of the timeframe in which biomarkers are incorporated into children’s growing hair so that appropriate adjustments can be made to hair biomarker levels associated with

environmental exposures and the mind-brain-body connectome. MATERIALS AND METHODS STUDY COHORT Following approval from the Stanford University IRB, parents or guardians gave informed consent

for participation in the Hair Biomarkers Study (https://childwellness.stanford.edu/hair-biomarkers-study). Healthy preschool children aged 9–72 months (_N_ = 266) from Santa Clara County,

CA were enrolled in a 3-week longitudinal observational study to evaluate HGRs during the period of November 2017 to February 2020. Subjects with tinea capitis, alopecia areata, eczema, or

other scalp conditions, or those receiving any prescription or other drugs (except daily vitamins) were excluded. Subjects receiving systemic steroid therapy, or those with a history of

chronic medical conditions such as cystic fibrosis, sickle cell disease, congenital heart disease, or other disorders, and subjects with chemical exposures (e.g., dying, bleaching, chemical

straightening, perming) to their hair in the 3 months prior to study entry were also excluded. Qualitative assessment of social and demographic factors confirmed that our study sample only

included healthy children from nuclear families free from adversity. The COVID-19 pandemic limited the enrollment and in-person contact with several participants for serial HGR measurements.

DETERMINANTS OF HAIR GROWTH Hair samples, demographic information, and anthropometric measures were collected at seven participating sites. Parents were asked to classify their child’s race

into racial categories recognized by the Federal government (White, Asian, African American, Alaska Native, or Pacific Islander). Ethnicity was assigned in accordance with the Department of

Homeland Security’s code of ethnicities (based on the geographic location of birth, namely, Africa, Asia, Caribbean, Central America, Europe, North America, Oceania, and South America).

Subjects were designated as “Mixed” if maternal and paternal race or ethnicity differed, assigned to the “Other” race group if they were identified as African American, Native Alaskan, or

Pacific Islander, and into the “Other” ethnicity group if they were identified as African, Caribbean, or Oceanian. To assess nutritional status, we measured the child’s height, head, and

waist circumferences. We classified children into three hair pigment groups (light, medium, and dark hair) using a modification of Loussouarn’s methodology, which involved matching hair

color to a reference scale compromised of a 10-unit gradient ranging from black (1) to pale blonde (10).27 Dark pigmentation included black/brown, black and dark black hair colors (pigments

1–3). Medium pigmentation included brown/red, light brown, brown and dark brown hair colors (pigments 4–7). Light pigmentation included gradients from pale blonde, blonde/brown and

blonde/red hair colors (pigments 8–10). Hair sampling seasons (Spring, Summer, Autumn, Winter) were identified by dates of the two solstices and two equinoxes to assess seasonal changes in

hair growth. HAIR SAMPLING Researchers were trained to trim a 1 cm2 area near the posterior vertex using a Philips Norelco Multigroom 3000 trimmer®, which cuts hair at 0.1 mm from the scalp.

Fine digital calipers (Fisher Scientific) were used to measure the length of new hair at weekly follow-up visits. The same researcher who obtained hair measured the hair growth at weekly

appointments across 7–21 days. Hair growth was averaged based on serial measurements of 3–5 hairs. The difference in hair length from the previous measurement was divided by the number of

days between measurements to calculate the averaged hair growth rate (HGR) (μm/day). HAIR PROTEIN EXTRACTION AND MEASUREMENT The distal end of hair was taped and the proximal (scalp) end was

indicated by an arrow on a folded paper containing the sample. The hair sample was placed into a zip-lock bag, labeled, and stored at 4 °C until processing.12 The proximal 0–3 cm length of

hair was weighed (10–50 mg) in a glass vial, finely cut to a powdery consistency, and processed with a four-step extraction procedure, with alternating two cycles of hair extraction in 1 ml

of methanol (15 h, at 52 °C, rotating 200 rpm) followed by 1 ml acetone (5 min, at room temperature, rotating 200 rpm). Samples were centrifuged, supernatants removed and air dried at 4 °C.

Dried residue was reconstituted with phosphate buffered saline (70 μl/10 mg hair) with transfer to microcentrifuge tubes. Samples were cleaned by cold (4 °C) centrifugation, 12,000 rpm for

15 min, and the supernatant was transferred to fresh tubes. The soluble hair protein content (HPC) was measured by spectrophotometric absorption at 260/280 (Take-3, Epoch plate reader,

Gen5.5 software, BioTek Instruments). STATISTICAL METHODS Descriptive statistics were used to summarize demographic characteristics and anthropometric measures for children enrolled. These

variables were summarized using means with standard deviations or medians with interquartile ranges for continuous variables, frequencies and percentages for categorical variables. HGRs for

each follow-up visit were averaged to obtain an overall mean HGR and then summarized by age group, sex, race, height tertiles, age-adjusted height tertiles, hair pigmentation, and hair

sampling season. ANOVA and _post hoc_ two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli was performed to assess whether HGRs differed by age, sex, race, height,

age-adjusted height, hair pigment, or season. To examine associations between HGR and factors of interest, we performed several simple and multiple linear regression models. Simple linear

regression was used to model the bivariate relationship between HGR and each factor. Multiple linear regression was used to examine the association between HGR and all factors

simultaneously. Data were fitted to a linear regression model that included age, sex, race, height, hair pigmentation, and hair sampling season (Model 1) and HPC was added in Model 2.

Multiple imputations using fully conditional specification were used to impute any missing independent covariates used in these models. We created ten imputed datasets, and estimates were

combined using Rubin’s rules. The ‘type3_MI_glm’ macro developed by Wang et al. was used to generate p-values from type-III analyses.51 All analyses were conducted using SAS 9.4 (SAS

Institute, Inc., Cary, NC) and R (version 4.1.2.; R Foundation for Statistical Computing, Vienna, Austria). All tests were two-sided and evaluated using an alpha-error of 0.05. RESULTS Table

 1 shows child demographics and measurements for the study sample (_N_ = 266). Participants had an average age 3.5 ± 1.3 years, height 101.2 ± 12.2 cm, head circumference 49.7 ± 3.0 cm, and

waist circumference 51.2 ± 4.5 cm. Nearly half of the subjects (44.7%) were White, while the remaining subjects were Asian (25.2%), Mixed (16.5%), and Other (13.5%, including African

American, Pacific Islander, and Alaska Native). Most subjects belonged to non-Hispanic (88.7%) ethnicity. We studied similar numbers of male (54.1%) and female (45.9%) subjects and subjects

with light (27.8%), medium (39.5%), and dark (32.7%) hair. Table 2 shows the distribution of sex, race, hair pigment, and anthropometric measures for children within each age group

(<1-year-old, 1-, 2-, 3-, 4-, and 5-year-old children). HAIR GROWTH RATES The average HGR ranged from 287.9 ± 60.7 μm/day in infants less than 1 year old to 345.1 ± 47.2 μm/day in

3-year-old children. HGR rates differed among age groups, with infants less than 1 year old showing slower HGRs than 2-, 3-, 4-, and 5-year-old children (all _p_ < 0.05, Table 3). We

found no sex differences in HGRs (female 338.4 ± 47.2 μm/day vs. male 340.7 ± 50.4 μm/day; _p_ = 0.71). While HGR was not associated with waist or head or arm circumferences, subjects in the

top tertiles for height and age-adjusted height had higher HGRs than those in the bottom tertile (_p_ = 0.0025 and _p_ = 0.04, respectively, Table 3). Subjects with dark hair pigment (352.4

 ± 51.6 μm/day) had higher HGRs compared to subjects with light hair pigment (325.3 ± 50.0 μm/day; _p_ = 0.0019, Table 3). Asian subjects had the highest HGRs among all races and

significantly higher HGR compared to White subjects (355.6 ± 50.3 μm/day vs. 332.1 ± 48.5 μm/day, respectively, _p_ = 0.016, Table 3). HGRs differed across the age groups for White children

(_p_ = 0.0026) but not Asian children (_p_ = 0.19), and were inconclusive for “Mixed” and “Other” race groups. No HGR differences were observed between the four seasons. Table 4 presents the

results of linear regression analyses. Consistent with the findings from ANOVA in Table 3, simple linear regression showed significant relationships between HGR and age (_p_ = 0.0073),

height (_p_ = 0.0025), race (_p_ = 0.016) and hair pigmentation (_p_ = 0.0019). Age (_p_ = 0.0066) and height (_p_ = 0.005) remained significant when other factors were adjusted in the

linear regression model (Model 1). Model 1 explained 15% of the variation in children’s HGR. With addition of hair protein content (HPC) to the model (Model 2), significant differences in

HGR were seen for age (_p_ = 0.018), height (_p_ = 0.0073) and HPC (_p_ = 0.013), whereas hair pigmentation had marginal effects (_p_ = 0.062). Model 2 explained 17% of the variation in HGR

and fit the data better (_p_ = 0.01) as compared to Model 1. An R-squared of 0.17 corresponds to Cohen’s effect size F-squared of 0.20, which based on Cohen’s 1988 criteria, indicates a

medium effect size for HGR variability in children. We also tested the assumptions for both linear models. As shown in Fig. 1, the density plots and scatterplots of studentized residuals for

hair growth rates followed a linear pattern for both Models 1 and 2, showing that the linearity and the equal variance assumptions were being met.52 In addition, the residuals for both

models were not skewed, because all points in the Quantile-Quantile plots (Q-Q plots) were distributed along the 45-degree diagonal reference line, further substantiating that the normality

assumption was satisfied (Fig. 1).52 HPC was negatively correlated to HGR (_r_ = –0.18, _p_ = 0.001) and significantly altered by age, showing higher values in infants below 1 year compared

to children at older ages (_p_ = 0.0014, Fig. 2a). There were no differences in HPC between children grouped by race (_p_ = 0.2261). HGRs were lower in the youngest children and

significantly higher at 2–5 years (_p_ = 0.0073, Fig. 2b). Given these findings and improvement in the HGR regression model after adding HPC, we adjusted each child’s HGR by their measured

hair protein content. The ratio of HPC to HGR revealed the highest levels of hair protein synthesis below age 1 year with steady decline through to age 5 (_p_ = 0.0001, Fig. 2c). DISCUSSION

This is the first study to determine potential covariates of linear hair growth in preschool children. Linear HGRs in preschool children are age-dependent with overall increases in the HGR

as children age; HGRs are lowest in children under 1 year of age and highest in children aged 3–5 years. These findings are consistent with the findings of Barth and de Kruijff et al., who

reported lower HGRs in children below 2 years of age.38,42 Furthermore, age explains a moderate degree of HGR variance when comparing adult vs. child HGRs from Asian, European, and North

American groups (30, 39, and 55% variance, respectively; see Supplementary). There are several plausible explanations for the age-dependent differences in HGRs. First, newborn hair has much

less medulla than infant hair, and the degree of medullation, which is associated with the anagen phase of hair growth, increases throughout childhood.22 Furthermore, it is hypothesized that

the four types of infant hair (i.e., lanugo in newborns, vellus hair, an intermediate form of hair, followed by terminal hair) grow at different rates.53 Second, around the first year of

life the hair growth cycle transitions from a state of synchronous to asynchronous activity in the hair follicles.38,54 This transition results in variable hair loss and significant

differences in the percentage of terminal hair follicles; subsequently, there is a transient decrease in HGR around 1 year of age. Once hair growth has transitioned from synchronous to

asynchronous cycles, hairs are uniformly distributed to all stages of the growth cycle, which results in a relative net increase in HGR (when compared to the transition period) in older

children—this is maintained unless disrupted by ageing, hormonal changes, or illness.19,20,38,55,56,57 When comparing within ethnicities, child HGRs observed in our study differ from

published adult HGR ranges.27 The discrepancy between adult and child HGRs may promote inaccurate interpretations of biomarker levels; thus, we caution investigators applying adult linear

HGRs for defining the time-period of biomarker incorporation into children’s hair. For example, variations in linear HGRs (child vs. adult) may partially account for the higher hair cortisol

levels observed in young children as compared to adults.11,42,43 Our study also determined that race, hair pigmentation, height, and age-adjusted height are covariates of HGR in preschool

children. Specifically, HGRs were higher in Asian vs. White children, those with dark vs. light pigmented hair, and in the taller children. Examination of the age-dependent inverse

relationship between HGR and HPC (i.e., younger children have lower HGR and higher HPC) via the HPC to HGR ratio revealed that hair protein synthesis is highest in children below 1 year of

age and decreases as children get older. One plausible explanation for these findings may be that the protein utilization required for increased height growth velocity may lead to lower HPC

in older/taller children, although further research directly comparing the rates of total body protein synthesis with hair protein synthesis will be required to assess this relationship. In

adults, decreases in hair pigmentation are associated with decreases in telogen (resting phase) density, with likely increases in the proportion of anagen hair follicles in adults with

lighter color hair.27 Longitudinal analyses indicate that cycling of hair follicles is independent of linear HGRs in adults.56 Therefore, even if children with light-colored hair had a

higher proportion of anagen hairs, that does not contradict our finding of lower HGRs in these children. There is limited data on the effect of malnutrition on hair growth rates in preschool

children.58 Our findings did not indicate an association between waist circumference and HGR. However, our study protocol included a healthy population at low risk of malnutrition. No other

published data on anthropometric measures and HGRs was identified, therefore, future studies enrolling at-risk populations may provide insight on the undetermined association of HGR and

other anthropometric measures in preschool children. Strengths of this study include a larger and more diverse sample than all prior studies on hair growth. We also studied a broad range of

potential covariates and identified several HGR-related factors in preschool children. Thus, our study provides determinants of linear HGRs for previously understudied preschool children.

Weaknesses of this study include the uneven distribution of participants from different racial/ethnic groups. Very few subjects identified as Pacific Islander, Alaska Native, or African

American; these subjects were collectively assigned to the “Other” group. Uneven distribution of boys and girls also occurred in the <1-year age group, though HGRs did not differ across

sex in this or other age groups. Lack of hair quality assessment (given the established racial and ethnic differences in hair qualities, such as texture, form, and thickness) was another

weakness of our study.27,35,38,59,60 We advise that future reference studies must include diverse populations and incorporate objective metrics for evaluating hair qualities, so that

meaningful associations between hair growth and race/ethnicity can be investigated to facilitate the interpretation of hair biomarkers in children. CONCLUSIONS Compared to the existing

literature on linear hair growth in children, our data provides potential covariates of hair growth in a racially and ethnically diverse population of preschool children. Determinants of

linear HGR in preschool children will be essential to implement biomarker testing and to accurately interpret their results in pediatric research and clinical practice. Our study reveals

age, race, hair pigment (color), and height as covariates of HGRs in preschool children; studies measuring hair biomarkers should account for these determinants. Moreover, our initial

observations on hair protein content and HGRs may provide physiological explanations for differences in HGRs and biomarkers in preschool children as compared to adults. DATA AVAILABILITY The

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thank Grace K-Y. Tam, Clinical Research Coordinator, Pain/Stress Neurobiology Laboratory, and Dr. Sukyung Chung, Quantitative Sciences Unit, Stanford University School of Medicine, Stanford,

CA for their contributions to this study and to earlier versions of this manuscript. FUNDING Grants from the _Eunice Kennedy Shriver_ National Institute for Child Health & Human

Development (R01 HD099296) and the Maternal & Child Health Research Institute to KJSA supported this study. Study sponsors had no role in the design and conduct of the study; the

collection, management, analysis, or interpretation of the data; the preparation, review, approval, or decision to publish this manuscript. AUTHOR INFORMATION Author notes * Mónica O. Ruiz

Present address: Department of Pediatrics, Brown University School of Medicine, Rhode Island Hospital & Hasbro Children’s Hospital, Providence, RI, USA AUTHORS AND AFFILIATIONS *

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Mónica O. Ruiz, Cynthia R. Rovnaghi & Kanwaljeet J. S. Anand * Stanford Child Wellness Lab, Maternal

& Child Health Research Institute, Stanford, CA, USA Mónica O. Ruiz, Cynthia R. Rovnaghi, Sahil Tembulkar, Leni Truong & Kanwaljeet J. S. Anand * Quantitative Sciences Unit, Stanford

University School of Medicine, Stanford, CA, USA FeiFei Qin & Sa Shen * Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University School of Medicine, Stanford,

CA, USA Kanwaljeet J. S. Anand Authors * Mónica O. Ruiz View author publications You can also search for this author inPubMed Google Scholar * Cynthia R. Rovnaghi View author publications

You can also search for this author inPubMed Google Scholar * Sahil Tembulkar View author publications You can also search for this author inPubMed Google Scholar * FeiFei Qin View author

publications You can also search for this author inPubMed Google Scholar * Leni Truong View author publications You can also search for this author inPubMed Google Scholar * Sa Shen View

author publications You can also search for this author inPubMed Google Scholar * Kanwaljeet J. S. Anand View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS M.O.R.: substantial contributions to data analysis and interpretation; drafting the article and revising it critically for important intellectual content; and final approval of

the version to be published. C.R.R.: substantial contributions to conception and design; data acquisition, analysis, and interpretation; drafting the article and revising it critically for

important intellectual content; and final approval of the version to be published. S.T.: substantial contributions to conception and design; acquisition of data; and final approval of the

version to be published. F.Q.: substantial contributions to data analysis and interpretation; and final approval of the version to be published. S.S.: substantial contributions to data

analysis and interpretation; and final approval of the version to be published. L.T.: substantial contributions to data acquisition and analysis; and final approval of the version to be

published. K.J.S.A.: Substantial contributions to conception and design; data acquisition, analysis and interpretation; drafting the article and revising it critically for important

intellectual content; securing grant funding for the project; and final approval of the version to be published. CORRESPONDING AUTHOR Correspondence to Mónica O. Ruiz. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Parents/Guardians of child participants gave written consent to trained research

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terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ruiz, M.O., Rovnaghi, C.R., Tembulkar, S. _et al._ Linear hair growth

rates in preschool children. _Pediatr Res_ 95, 359–366 (2024). https://doi.org/10.1038/s41390-023-02791-z Download citation * Received: 13 September 2022 * Revised: 13 July 2023 * Accepted:

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