Changing

Article Title Hormone Levels During the

Menstrual
Article SubtitleCycle Affect Knee Laxity and
Stiffness
Author in Healthy Female Subjects
Affiliation
Sang-Kyoon Park,* PhD, Darren J. Stefanyshyn,* PhD, Barbara Loitz-Ramage, PT, PhD,

David A. Hart, PhD, and Janet L. Ronsky,* PhD

From the *Human
The abstract Performance
goes here and Laboratory, Faculty of Kinesiology, the McCaig Institute
covers two columns.

for Bone & Joint Health, Faculty of Medicine, and the Department of Mechanical
The abstract goes here and covers two columns.
and Manufacturing Engineering, Schulich School of Engineering, University of Calgary,
The abstract goes here and covers two columns.
Calgary, Alberta, Canada
The abstract goes here and covers two columns.

KEY WORDS list of key words goes here

Background: Whether knee laxity varies throughout the menstrual cycle remains controversial. As increased laxity may be a risk
factor for anterior cruciate ligament (ACL) injury, further research is warranted.
Hypothesis: Variation in estradiol and progesterone levels during the menstrual cycle influences knee laxity and stiffness.
Study Design: Case control study; Level of evidence, 3.
Methods: The serum estradiol and progesterone levels of 26 healthy female subjects were recorded in the follicular phase, ovu-
lation, and the luteal phase. Knee joint laxity was assessed using a standard knee arthrometer at the same intervals. Stiffness
changes in the load-displacement curve were determined. Hormone levels across the cycle were compared between responders
and nonresponders, defined by whether changes in knee laxity at 89 N occurred.
Results: Greater laxity at 89 N during ovulation was observed (ovulation: 5.13 1.70 mm vs luteal: 4.55 1.54 mm, P = .012).
In knee laxity testing at manual maximum load, greater laxity was noticed during ovulation (14.43 2.60 mm, P = .018), as com-
pared with the follicular phase (13.35 2.53 mm). A reduction in knee stiffness of approximately 17% (ovulation: 12.48 5.46
N/mm vs luteal: 15.02 7.71 N/mm, P = .042) during ovulation was observed. However, there were no differences in hormone
levels between responders and nonresponders at 89 N.
Conclusion: Female hormone levels are related to increased knee joint laxity and decreased stiffness at ovulation. To understand
subject variations in knee joint laxity during the menstrual cycle in female athletes, further investigation is warranted.
Keywords: estradiol; progesterone; responders; nonresponders; ACL injury

During sports activities, female athletes tear their anterior movement between male and female athletes may also
cruciate ligament (ACL) 2 to 8 times more frequently than account for the varying incidence. For example, female sub-
their male counterparts,3,30 prompting an important research jects show excessive pronation with increased tibial rotation
question about why such differences might exist. Structural during running,25 and greater knee valgus and less knee
differences between male and female athletes, such as a flexion angles during a cutting maneuver and landing.11,15,41,43
greater Q angle,30 greater patellar tendontibia shaft angle,50 Moreover, sex-related differences in joint-loading patterns
and narrower femoral intercondylar notch,60 have been sug- exist, with female subjects exhibiting greater knee internal
gested as anatomical factors that may contribute to an adduction moments during cutting and landing.11 Isokinetic
increased injury rate among women. Different patterns of strength testing has shown weaker quadriceps and ham-
string muscles in female athletes compared with their male
Presented at the interim meeting of the AOSSM, San Francisco, counterparts, even when data were normalized to subject
AddressMarch
California, correspondence
2001. to Darren J. Stefanyshyn, PhD, Human
Performance Laboratory, University of Calgary, 2500 mass.31 Also, decreased neuromuscular characteristics, such
Address correspondence to Author 1, Address 1, University
City, State,Drive,
Zip,
NW, Calgary, Alberta T2N 1N4, Canada (e-mail: darren@kin.ucalgary.ca).
Affiliation. as decreased joint proprioception and a delayed muscle
No
Anypotential conflict
authors notes of interest
could also go declared.
here. reflex, may cause a diminished protective mechanism to the
The
external loads.59 It has been speculated that a greater ante-
The American
American Journal
Journal of
of Sports
Sports Medicine,
Medicine, Vol.
Vol. 37,
33, No.
No. 3X rior knee laxity with a quadriceps dominant pattern in
DOI:
DOI: 10.1177/0363546508326713
10.1177/1073858403253460

2009
2005 American
American Orthopaedic
Orthopaedic Society
Society for
for Sports
Sports Medicine
Medicine females is likely related to ACL injury risk.31

588
Vol. 37, No. 3, 2009 Hormone Levels Affect Knee Laxity and Stiffness 589
Most of the previous studies have focused on the different
ACL injury mechanics between men and women.|| Few stud-
ies have examined within-gender differences in injury
mechanism,17,48,62 with no work addressing why some
female athletes suffer ACL injures while others do not.
Recently, studies have pointed to greater joint laxity as a
potential contributor to higher ACL injury rates among
female athletes,18,26,51,62,63,65,83 with some researchers report-
ing that hormones significantly influence joint laxity
throughout the menstrual cycle.18,21,26,63 Research indicates
that increased knee joint laxity can be observed during (pre)
ovulation (days 10-14).18,21,26,63 Also, a relationship appears
to exist between the frequency of ACL injuries and hormone
levels during the menstrual cycle.2,28,46,47,49,65,76,77
Greater knee joint laxity may negatively affect dynamic
joint function in female athletes due to corresponding
decreases in both the preactivation of muscles to prepare
the joint for the application of external forces8,74 and control
of joint position in space.38,59,61 As a result, female athletes
may be more prone to injury during specific times of their
menstrual cycle because of greater joint laxity. However,
recent studies examining a relationship between hormones
and knee joint laxity during the menstrual cycle remain
equivocal. Five studies reported an increase in knee laxity
during ovulation (10-14 days),18,21,26,62,83 while others found
no significant hormone-related effect on knee joint
laxity.4,7,9,33,39,58,71 This discrepancy may result from study
design limitations,83 such as using more than 1 examiner for
laxity measurements,9,62,63 or recruiting inappropriate con-
trol subjects, such as women on oral contraceptives or pre-
pubertal girls for the subject population.4,33 Also, although
serum analysis of hormone concentrations has been shown
to be a more reliable and accurate method of determining
the cycle phase,83 some studies have used self-reported
questionnaires26,33 or self-assessments of basal body
temperature7,18 rather than serum analysis. Moreover, only
a few studies have used an ovulation kit with serum sam-
ples to detect the right timing of ovulation.9,58,71 In a recent
extensive systematic review using meta-analysis, Zazulak
et al83 suggested that the hormonal cycle has an effect on
knee joint laxity. However, they also proposed that further
investigation is required to confirm their conclusion because
of the limitations in most previous studies.83 Thus, any
future investigators should consider the noted limitations
and control for the many confounding factors to understand
the potential links between the ACL injury mechanism and
knee joint laxity during the menstrual cycle.
Only a few studies report a joint-stiffness parameter,
which is more indicative of the tensile properties of tissue
than joint laxity taken at certain loads.18,20,39,58 Previous
studies computed linear stiffness between arbitrarily cho-
sen loads (45, 89, and 133 N),18,58 possibly missing impor-
tant data, as stiffness changes are subject dependent and
linear stiffness may not fit well with a given loading regi-
men. Assessment of variations in stiffness at different
loads would be an appropriate approach to quantify the
experiences of individual subsets. As a result, most studies
||
References 11, 15, 19, 23, 41, 43, 44, 54, 78, 81, 84.
References 4, 7, 9, 18, 21, 26, 33, 39, 58, 62, 63, 71.
only reported knee joint laxity. An exception to this is a
study that reported knee stiffness from a standard
arthrometer; it showed disagreement between the knee
laxity and stiffness measures.18 Thus, a more appropriate
calculation of ligament stiffness appears warranted to
understand the relationship between hormonal cycle and
structural behavior of the knee.
Hormone level changes and their contribution to knee
joint laxity are known to vary between subjects.62 However,
most current studies do not explain why some subjects are
influenced by changing hormone levels while others are
not.62,63 Subject variations in hormone levels may be a
strong confounding factor to hide a possible relationship
between knee joint laxity and the menstrual cycle.62,83
Therefore, careful observation of individual hormone levels
could explain subject variability in knee laxity. Thus, the
objectives of this study were to determine (1) whether
changing hormone levels influence joint laxity and stiffness
of the noncontractile knee joint structures using a new
analysis technique and (2) whether subsets of women exist
who demonstrate (responders) or do not demonstrate (non-
responders) changes in knee laxity in response to circulat-
ing hormone levels throughout their menstrual cycle. The
hypothesis was that knee laxity increases from the follicu-
lar phase to ovulation due to the effect of high estradiol on
the ligament during ovulation and decreases from ovula-
tion to the luteal phase because of high progesterone levels.
The second hypothesis was that absolute hormone levels
differed between responders and nonresponders.
MATERIALS AND METHODS
Each subject completed a blood draw and laxity tests at 3
different times during her menstrual cycle. Blood samples
were collected to determine the levels of estradiol and pro-
gesterone, indicating an appropriate phase of testing.
Passive knee laxity and stiffness were measured using a
standard arthrometer. Data were processed to investigate
whether hormone levels affect knee laxity and stiffness
across the menstrual cycle.
Subject Inclusion Criteria
Twenty-six young women were recruited, and their charac-
teristics were as follows: age, 22.7 3.3 years; height, 170.1
7.1 cm; mass, 65.0 9.3 kg; body mass index (BMI), 22.4
2.5; average menstrual cycle, 28.9 2.7 days; and activity
level, 8.7 4.4 h/wk. Most subjects regularly participated in
a sports activity at a recreational level. Most subjects (20 of
26) regularly participated in a sports activity associated
with running, jumping, and cutting maneuvers (running, 5;
basketball, 4; soccer and rugby, 3; volleyball, 3; gymnastics,
2; dance, 2; squash, 1), while the other 6 subjects partici-
pated in other sports activities (swimming, 2; biking, 1;
wrestling, 1; karate, 1; figure skating, 1). Inclusion criteria
were no previous knee injuries, never been pregnant, have
regular menstrual cycles (approximately 28 days)73 with no
missed cycles over the previous 24 months, and have had
no oral contraceptive use for the previous 6 months. Each
590 Park et al The American Journal of Sports Medicine
subjects average menstrual cycle was self-reported based
on the previous 3 months. The subjects completed a written
consent form approved by the institutional ethics review
committee. The first meeting focused on discussion of each
subjects menstrual history and an introduction to the test-
ing equipment and protocol.
Experimental Protocol (3 Measurements)
Each subject came to the laboratory 3 times, based on the
phases of her menstrual cycle. The 3 testing times were
determined as follows. The first measurements (follicular
phase) were taken 3 to 7 days after the beginning of the
menstrual cycle, when estrogen and progesterone levels
were expected to be low.26,63 The second data collection coin-
cided with ovulation and occurred 24 to 48 hours after the
estrogen surge detected by an ovulation predictor kit
(Clearblue, Bedford, UK) with 99% accuracy.57 The subjects
were given an ovulation predictor kit for home use and were
instructed when to employ the predictor kit based on their
menstrual history. For example, for a subject with an aver-
age 28-day menstrual cycle tested on days 11 to 15 of her
cycle, ovulation testing was done at the same time of day;
the subject had not urinated for at least 4 hours before test-
ing and avoided excessive fluid intake before testing. The
procedure involved holding a test stick in the urine stream
for 5 seconds or collecting the urine in a paper cup and dip-
ping the test stick into the cup for 20 seconds. When a posi-
tive result occurred, as indicated by a colored band on the
test stick, the subject contacted the primary investigator to
schedule data collection within the subsequent 48 hours.
The third data collection (luteal phase) occurred approxi-
mately 7 days after ovulation (based on an average 28-day
menstrual cycle) and was indicative of peak progesterone
levels.33,37 Figure 1 illustrates the 3 times the hormone level
testing was performed during a single menstrual cycle.
Blood Draw
Two certified and experienced phlebotomists, with 2 and
10 years experience, performed each venipuncture. The
samples were collected from the antecubital area of the
subjects arm, using a 22-gauge needle and a 3.5-mL
Vacutainer serum separator tube (BD, Franklin Lakes,
New Jersey). Once the tube was completely filled, the
sample was mixed well and allowed to clot for 20 minutes.
The serum was then immediately separated by centrifuga-
tion in a Sorvall Legend RT centrifuge (Thermo Fisher
Scientific, Waltham, Massachusetts), set at 3000 rpm for
10 minutes. The 1 mL of serum was then carefully decanted
into a labeled 5-mL culture tube, refrigerated, and sent to
the local clinical laboratory service (Calgary Laboratory
Service, Calgary Health Region) for progesterone and
estradiol analysis.
Knee Laxity Measurement
The KT-2000 arthrometer (MEDmetric Corp, San Diego,
California) was used to quantify anterior tibial displacement
Figure 1. Chart indicating the timing of hormone level
testing during the menstrual cycle. #1, follicular phase; #2,
ovulation; #3, luteal phase.22 Modified with permission from
Slauterbeck et al.65
and thus to infer ACL laxity. One researcher performed all
the arthrometer measurements to eliminate interrater
differences in the laxity measure. Each subject restrained
from excessive fluid intake for at least 1 to 2 hours before
laxity testing. Subjects were positioned supine with
their legs resting on a thigh support in 30 of knee flexion
to ensure uniform test-retest flexion angles. A foot support
platform used during testing maintained consistent hip
abduction and rotation positions. The KT-2000 arthrometer
was placed on the anterior aspect of the right tibia and
firmly secured proximally and distally with circumferential
straps. Because a right-handed examiner is more reliable
in testing the right leg of a subject during laxity measure-
ment, only the right knee was tested.71
The average of 3 trials of a passive drawer test was ana-
lyzed for each subject. Each trial included displacement loads
at 89 N applied through a force handle located 10 cm distal
to the joint line of the knee. Additionally, a separate manual
maximum displacement test was performed by applying a
strong anterior displacement force directly to the subjects
proximal calf with one hand to produce the maximum ante-
rior displacement, while the patellar sensor pad was stabi-
lized with the other hand.55 The maximum anterior tibial
translation was recorded from the displacement dial when
the needle stopped moving. The average of 3 trials of manual
maximum testing was analyzed for further comparison.
The KT-2000 arthrometer recorded ASCII data that
enabled the calculation of an approximate linear stiffness
from the slope of a straight line fitted to sections of the
load-displacement curve. Three distinct stiffness or slope
changes were observed from the first derivative during the
ascending limb of the knee laxity curve (Figure 2). To iden-
tify dramatic slope changes (inflection points) on the curve,
the second derivative of the displacement-load curve was
applied. Local minima and maxima points were defined
by the peak of the second derivative on the graph (Figure
2). This procedure allows calculation of stiffness in the 3
regions of the load-displacement curve: (1) the initial rise
as the weight of the leg is overcome, causing a relatively
steep slope; (2) the middle region as slack is taken up in
the passive joint structures; and (3) the linear region where
the onset of primary restraint to anterior tibial translation
occurs.40 The first stiffness was calculated from the slope
Vol. 37, No. 3, 2009 Hormone Levels Affect Knee Laxity and Stiffness 591
Figure 2. Calculation of the local minima and maxima
points of the second derivative of the joint laxity curve. S1,
stiffness from the origin to the local minima point; S2, stiff-
ness from the local minima point to the local maxima point;
S3, stiffness from the local maxima point to the end of data;
solid line, the load-displacement curve; dashed line, the
first derivative of the curve; dotted line, the second deriva-
tive of the curve.
between the origin and the local minima point, the second
stiffness was calculated from the slope between the local
minima point and the local maxima point, and the third
stiffness was calculated between the local maxima point
and the end point of the load-displacement curve (Figure
2). Data were filtered using a low-pass Butterworth filter
at a cutoff frequency of 30 Hz. This cutoff frequency was
defined using a power spectrum analysis of both force and
displacement data, based on a fast Fourier transform.69
The simple linear regression equation was computed for
3 different stiffness regions based on calculated local
minima and maxima points, with each linear slope being
provided by Matlab, version 6.5.1 (Math Works Inc, Natick,
Massachusetts).
Repeatability of Knee Laxity Measurement
The intratester repeatability of the KT-2000 arthrometer
was determined by an intraclass correlation coefficient
(ICC)68 of laxity measured from 7 of the subjects on 2 con-
secutive days. Standard error of measurement (SEM) was
calculated to determine the measurement precision and
reliability analysis, and the Cronbach model was used to
evaluate the extent of agreement between the consecutive
tests. The initial laxity testing was performed with the
same protocol used for the detailed experiment. The second
test was carried out the next day at the same time of day
(half an hour). Repeatability of displacement between the
measurements was given by an ICC (3,2) of 0.98 for laxity
at 89 N (F6,6.0 = 75.42, SEM = 0.37 mm, P < .001) (first test:
6.23 1.52 mm vs second test: 6.27 2.36 mm) and 0.91 for
maximum manual laxity test (F6,6.0 = 23.135, SEM = 1.10
mm, P < .001) (first test: 14.82 2.54 mm vs second test:
14.93 2.63 mm). Thus, the tester showed reliable repeat-
ability at 89 N and manual maximum test over time.
Responders and Nonresponders
Subjects were divided into responders and nonresponders
depending on whether changes in knee laxity at 89 N were
detected from the follicular phase to ovulation and from ovu-
lation to the luteal phase. The threshold to determine the
response types was defined as 0.4 mm based on the average
variability (between 0.35 and 0.4 mm) computed from the
repeatability data previously described. Subjects who showed
a laxity increase greater than the defined threshold (0.4 mm)
from the follicular phase to ovulation were defined as ovula-
tion responders, while the others were defined as ovulation
nonresponders. Subjects who showed a decrease in laxity
greater than the defined threshold (0.4 mm) from ovulation
to the luteal phase were identified as luteal responders,
while the others were defined as luteal nonresponders.
Group differences in hormone levels were then compared.
Statistical Analysis
At 3 times during the subjects menstrual cycles, the influ-
ence of female hormones on knee joint laxity and stiffness
was analyzed using a 1-way repeated measures analysis of
variance followed by a paired t test with a Bonferroni
adjustment for multiple comparisons (the differences
between the 2 different phases; the P value was multiplied
by the number of comparisons [n = 3]). A 2-way repeated
measure analysis of variance determined whether there
was a phase (time) effect, group effect, or phase-by-group
effect in laxity and hormone levels. To evaluate the new
method of computing stiffness compared with the typical
stiffness calculation, an R2 value was computed from a
simple linear regression analysis of the load-displacement
curves. Statistical analysis was performed using SPSS for
Windows, v.14.0, at an of .05 (SPSS Inc, Chicago, Illinois).
RESULTS
Hormone Levels
Estradiol and progesterone levels differed significantly
across the 3 testing sessions (P = .000) (Table 1). The low-
est estradiol (44.49 23.77 pg/mL) and progesterone (0.99
0.46 ng/mL) levels were found at the follicular phase, and
the highest estradiol (137.49 85.82 pg/mL) and proges-
terone (11.43 7.01 ng/mL) levels were found during the
luteal phase (Table 1).
Knee Joint Laxity
Over the menstrual cycle, greater knee laxity at 89 N was
recorded during ovulation compared to the luteal phase.
Knee laxity at 89 N decreased 11.3% from ovulation (5.13
1.70 mm) to the luteal phase (4.55 1.54 mm, P = .012)
(Figure 3). Maximum knee laxity during ovulation (14.43
2.60 mm, P = .018) significantly exceeded maximum laxity
during the follicular phase (13.35 2.53 mm) (Figure 3).
592 Park et al The American Journal of Sports Medicine

TABLE 1
Summary of Hormone Levels at 3 Test Intervals in All Subjectsa

Bonferroni-Adjusted P Value
Follicular Phase, Ovulation, Luteal Phase, Overall Phase
Outcome Measure mean (SD) mean (SD) mean (SD) Effect, F(df) [P] F vs O O vs L F vs L

Days of test, d 6.12 (1.40) 16.08 (2.99) 22.85 (3.22) 343.277(2,50) [.000**] .000** .000** .000**
Estradiol, pg/mL 44.49 (23.77) 80.84 (40.49) 137.49 (85.82) 19.103(2,50) [.000**] .000** .012* .000**
Progesterone, ng/mL 0.99 (0.46) 3.24 (2.37) 11.43 (7.01) 45.952(2,50) [.000**] .000** .000** .000**
a
F, follicular phase; O, ovulation; L, luteal phase. One-way repeated measures analysis of variance was performed at = .05. Independent
2-tailed paired t test with a Bonferroni adjustment for multiple comparisons (P was multiplied by the number of comparisons [n = 3]) was
performed at = .05.
*P < .05. **P < .001.

Knee Joint Stiffness

Using data from all subjects, a paired t test with a Bonferroni
adjustment demonstrated that slope of the displacement-
force curve between the local minimum and maximum points
increased significantly from ovulation (12.48 5.46 N/mm) to
the luteal phase (15.02 7.71 N/mm, P = .042) (Figure 4).
There was a significant difference in the maxima point from
the second derivative of the curve between 4.58 1.57 mm
during ovulation and 4.23 1.46 mm during the luteal phase
(P = .048) (Table 2). The current stiffness calculation showed
greater R2 values in the linear stiffness compared to a typical
stiffness calculation (Appendix).

Subject Variability in Knee Joint

Laxity During the Menstrual Cycle
Thirteen of 26 subjects showed the least knee joint laxity
during the follicular phase. Of the remaining subjects,
3 were least lax during ovulation and 10 during the luteal
phase. Fourteen of 26 subjects displayed the greatest lax-
ity during ovulation compared with 10 subjects during
the follicular phase and 2 subjects during the luteal
phase.
Figure 3. Mean (SD) laxity at the 3 test intervals (follicular,
ovulation, luteal). DIS89, displacement at 89 N of the load-
displacement curve; MAX, displacement at manual maximum
load. *P < .05.
DISCUSSION
Effects of Hormone Levels on Knee Joint Laxity

Responders and Nonresponders
On average, greater knee laxity at 89 N was found dur-
ing ovulation than during the luteal phase; however, this
was not true for all subjects. Therefore, subjects were
divided into responders and nonresponders. Fourteen
subjects (responders) demonstrated at least a 0.4-mm
decrease in laxity at 89 N from ovulation to the luteal
phase and were therefore identified as luteal responders.
Twelve subjects showed less than 0.4 mm of decreased or
increased laxity over the same intervals and were thus
identified as luteal nonresponders. However, there were
no significant differences in hormone levels between the
responder and nonresponder subsets, meaning there was
no group effect.
Several researchers have speculated that increased knee
joint laxity during the menstrual cycle may be related to a
high ACL injury rate in female athletes.28,63,83 However,
before investigating the ACL injury mechanisms associ-
ated with the menstrual cycle, the relationship between
knee joint laxity and hormone levels must be clarified
because of contradictory results in this area. Zazulak
et al83 have pointed out several limitations in existing
studies investigating the relationship between the hor-
mone cycle and knee joint laxity. Their study notes 3 major
limitations:
1. A small sample size: For a power of .8, we esti-
mated that 22 subjects are required to detect a
0.6-mm difference in knee joint laxity at 89 N
Vol. 37, No. 3, 2009 Hormone Levels Affect Knee Laxity and Stiffness 593

Figure 4. (A) Differences in stiffness between ovulation and the luteal phase in the force-displacement curve. O, ovulation; L,
luteal phase; S(O), stiffness between minima and maxima points during ovulation; S(L), stiffness between minima and maxima
points during the luteal phase. *P < .05. (B) Differences in stiffness between ovulation and the luteal phase in the force-
displacement curve. O, ovulation; L, luteal phase; S(O), stiffness between 45 N and 89 N during ovulation; S(L), stiffness between
45 N and 89 N during the luteal phase.

TABLE 2
Summary of Knee Stiffness Data at 3 Test Intervals in All Subjectsa

Bonferroni-Adjusted P Value
Follicular Phase, Ovulation, Luteal Phase, Overall Phase
Outcome Measure mean (SD) mean (SD) mean (SD) Effect, F(df) [P] F vs O O vs L F vs L

Displacement 1, mm 1.16 (0.42) 1.21 (0.51) 1.10 (0.61) 0.633(2,50) [.535] 1.000 .861 1.000
Displacement 2, mm 4.17 (1.22) 4.58 (1.57) 4.23 (1.46) 2.437(2,50) [.098] .261 .048b 1.000
Load 1, N 31.80 (15.16) 30.80 (11.82) 31.24 (13.62) 0.046(2,50) [.955] 1.000 1.000 1.000
Load 2, N 71.07 (22.61) 71.58 (20.96) 76.93 (22.26) 1.007(2,50) [.373] 1.000 .618 .879
Stiffness 1, N/mm 27.49 (10.38) 28.50 (14.80) 34.80 (27.71) 2.206(2,50) [.121] .701 .288 .411
Stiffness 2, N/mm 14.04 (8.19) 12.48 (5.46) 15.02 (7.71) 2.548(2,50) [.088] .507 .042b 1.000
Stiffness 3, N/mm 40.96 (15.15) 43.76 (21.29) 39.82 (11.75) 0.694(2,50) [.504] 1.000 1.000 1.000
a
F, follicular phase; O, ovulation; L, luteal phase; displacement 1, displacement at the local minima point; displacement 2, displacement at
the local maxima point; load 1, load at the local minima point; load 2, load at the local maxima point; stiffness 1, slope from the origin to
the local minima point; stiffness 2, slope from the local minima point to the local maxima point; stiffness 3, slope from the local maxima
point to the end of data. One-way repeated measures analysis of variance was performed at = .05. Independent 2-tailed paired t test with
a Bonferroni adjustment for multiple comparisons was performed at = .05.
b
P < .05.

between the different phases of the menstrual
cycle.18 However, only 3 of 10 studies used at least
22 subjects in their investigations.4,33,63
2. Inconsistent subject criteria: Subject populations
are often nonhomogeneous with respect to activity
levels (ie, whether their subjects are athletes or
nonathletes),7,9,18 and there is no history of oral
contraceptive use.4
3. Testing method: Studies have not been well designed
to clearly document the phases of the menstrual
cycle.83 Collecting a serum sample is required for
accurate timing rather than counting days based
on the average length of the subjects cycles.83 Use
of ovulation predictor kits with hormone analysis
enables confirmation of the right timing of ovula-
tion, but only a few studies have used both analyses
to detect ovulation periods.9,58,71
While 5 of 10 studies have found an increase in knee lax-
ity near ovulation,18,21,26,62,83 others have found no signifi-
cant effect of hormone levels on knee joint laxity.4,7,9,33,39,58,71
However, 3 studies indicating significant differences in knee
laxity during the menstrual cycle also have been criticized
because of critical limitations of study design such as small
sample size21,26 and lack of hormone data.18
The purpose of this study was to determine whether
changing hormone levels influence knee laxity during the
menstrual cycle. The data showed greater knee laxity dur-
ing ovulation of 12.7% (at 89 N) and 8% (at maximum
manual load) compared with the other phases. The results
are consistent with a few previous studies of the hormone
influences on knee joint laxity.18,21,26,63 Deie et al18 found
greater laxity at ovulation (5.3 0.7 mm) compared with the
follicular phase (4.7 0.8 mm) at 89 N in 16 female subjects,
and their laxity values are similar to the results from our
study. In addition, our results show an 11.3% (at 89 N)
decrease in knee joint laxity from ovulation to the luteal
phase, consistent with previous reports of a 4.3% decrease
in laxity at 89 N26,63 and a 7.4% to 10% decreased laxity at
133 N.63 It has been suggested that during the luteal phase,
the main effect of estradiol on the increase in knee laxity
would be mitigated by high progesterone levels.58 Shultz
594 Park et al The American Journal of Sports Medicine
et al63 collected 20 blood samples in each subject to analyze
hormone levels during 1 menstrual cycle and measured
knee laxity in 22 nonathletes. When the time delay (4-4.5
days) of hormone action on knee structure was considered,
a multiple regression analysis of hormone levels accurately
predicted changes in knee laxity.63 In a separate study,
Shultz et al62 suggested that individual variability in hor-
mone profiles accounted for subject variability in knee joint
laxity. Their results indicated that high estradiol and low
progesterone levels during the early follicular phase would
play an important role in variation of knee joint laxity
throughout the menstrual cycle.62
Conversely, several studies have reported no significant
effects of the hormone cycle on passive knee laxity in
women.4,7,9,33,39,58,71 Karageanes et al33 investigated the exis-
tence of a clinical association between passive knee laxity
and the menstrual cycle in 26 female athletes. They found
an average joint laxity of 4.98 mm in the follicular phase,
5.24 mm (4% increase) in the ovulatory phase, and 5.09 mm
(1.9% decrease) in the luteal phase at 89 N, but the differ-
ence was not significant. Arnold et al4 included a relatively
large sample size (41 uninjured athletes, 8 nonathletes, and
8 injured athletes) to examine the relationship between
serum relaxin and knee laxity; no differences in knee laxity
between the menstrual phases were found. Even though
these 2 studies included larger sample sizes compared with
the other studies, no significant relationship between hor-
mone levels and anterior knee laxity was observed. However,
there were important confounding factors in these studies,
including the method used to determine the phase of the
cycle4,33 and the use of an uncontrolled subject population.4
The former study estimated the phase of the menstrual
cycle based on information from subjects questionnaires,33
and relatively young subjects (15.7 1.0 years) were
recruited, introducing greater variability in hormone levels
because regular menstrual cycles may not have been firmly
established at this age.83 The knee laxity test was conducted
by 2 examiners in this study, potentially introducing error
related to intertester variability.33 The latter study was also
dependent on self-reporting to determine the use of oral
contraceptives, but 18% of subjects failed to provide infor-
mation.4 Thus, it is questionable whether the differences in
knee joint laxity observed at different phases of the men-
strual cycle were blurred by confounding factors in these
investigations despite relatively large sample sizes.
The current study has been designed to take into account
the major limitations shown in the previous studies, by
using a large sample size, controlled and homogeneous
subjects, and a reliable method to determine the cycle. The
findings showed greater knee laxity during ovulation com-
pared with the other phases and confirmed that hormone
levels have a significant influence on knee laxity across the
menstrual cycle in most women. However, it remains to be
determined whether these influences have a direct or an
indirect effect on the tissue.
Effects of Hormone Levels on Knee Joint Stiffness
Knee joint stiffness may be an important variable charac-
terizing the behavior of the bone-ligament-bone complex
obtained from knee laxity testings load-displacement
curve.79 Load is the independent variable, and displace-
ment is the dependent variable in the load displacement.79
At the first region of the curve, a relatively high slope is
observed as the applied load overcomes the weight of the
leg. After this, the joint is easily stretched by a small force
as seen with a decrease in slope. Finally, a linear region
with stiffer slope is observed as a continuous load is applied
to the joint until the ultimate load is reached.40 Generally,
these 3 distinct regions are reported in most clinical knee
laxity testing, but the transition (inflection points) between
the different regions of the load-displacement curve varies
among individuals.35
We used a new technique to determine the changes in
slope between the local minima and maxima of the second
derivative of the load-displacement curve. While all previous
studies used a linear regression to quantify the knee stiff-
ness within20,58 or at18 given loads (45, 89, and 133 N) in the
load-displacement curve, in this study, the first and second
derivatives of the curve were used to determine 3 slopes or
stiffness changes. Three distinct linear slope (stiffness) changes
from the first derivative of the laxity curve were found. The
results showed that stiffness changed between 30.80 N and
31.80 N and between 71.07 N and 76.93 N at the local min-
ima and maxima points of the second derivative rather than
at previously used values of 45 N and 89 N. In addition, the
current technique showed a better prediction of the linearity
of the data compared with traditional methods, and greater
R2 values in the linear stiffness equation were seen com-
pared to a typical stiffness calculation. Even small differences
in R2 values between the new and typical stiffness calcula-
tion are potentially important. For example, in the period
from ovulation to the luteal phase, a typical stiffness calcula-
tion reveals no significant changes in stiffness between 45-N
and 89-N loads (Figure 4B), while the new calculation of
stiffness showed significant changes in stiffness at the mid-
dle portion of the curve (Figure 4A). Thus, to quantify the
load-displacement behavior of the knee, laxity tests should
consider the individual shape of the load-displacement curve
rather than the shape within fixed regions only.
The second purpose of this study was to investigate
whether hormone levels influence knee joint stiffness dur-
ing the menstrual cycle. The current study detected an
increase in stiffness when progesterone levels peaked dur-
ing the luteal phase. The effect of estradiol may be dimin-
ished with increased progesterone levels,58 explaining the
increased stiffness (P = .042) (Table 2), followed by the
decreased laxity at 89 N (P = .012) (Figure 2) from ovula-
tion to the luteal phase. Romani et al58 correlated hormone
levels and the stiffness of the knee between 89 N and 133
N in 20 female subjects. A significant negative correlation
between estradiol concentration and knee joint stiffness
near ovulation was observed.58 On the other hand, despite
insignificant stiffness changes between the local maxima
point and the end point of the test (high stiffness region),
greater stiffness was noted during ovulation (ovulation:
43.76 21.29 N/mm vs follicular: 40.96 15.15 N/mm,
luteal: 39.82 11.75 N/mm) (Table 2). This possibly indi-
cates the achievement of the linear loading region at a
lower load than during other phases of the menstrual
Vol. 37, No. 3, 2009 Hormone Levels Affect Knee Laxity and Stiffness 595
cycle. If a ligament reaches the linear region earlier at
ovulation, earlier microfailure of the ligament might
occur.79 However, further study will be required to investi-
gate this speculation.
Different Response Types:
Responders and Nonresponders
Hormone level changes throughout the menstrual cycle
are known to have an effect on basal laxity.62 The female
participants in this study displayed varying degrees of
change in knee laxity in response to these hormone
changes. The heterogeneous response suggests that
unknown genetic factors that control tissue response to
hormones may exist. Thus, we can now begin to assess
the differences between knee laxity responders and
nonresponders.
It is important to observe each subjects cyclic hormone
variation to understand why some women experience sig-
nificant changes in knee laxity during the menstrual cycle
while others do not.62 It has been reported that the most
likely cause of changes in knee laxity in most women is
variation in estradiol levels59,63 from the follicular phase to
ovulation. Therefore, we expected to observe differences in
hormone levels, especially estradiol, between subjects who
responded with alterations in knee laxity and those who
did not. However, there were no significant differences in
estradiol and progesterone levels between hormone
responders and nonresponders. Several studies have
included the analysis of a larger number of hormones and
investigated the effects of the combination of estradiol,
progesterone, and testosterone on knee ligaments.63,82
Shultz et al63 suggested that there is a positive relation-
ship between knee laxity and levels of estradiol and
testosterone but that progesterone levels introduce sub-
ject-dependent changes in laxity. Thus, they suggested that
the role of each hormone and its interactions on ligament
response should be included to explain individual variabil-
ity in laxity.
With the present data set, the question regarding why
some individuals are responders and others are nonre-
sponders could not be addressed. Intrinsic and extrinsic
factors not considered in this study might influence a
womans relative sensitivity to hormone levels. For exam-
ple, individual body composition,32 menstrual history (con-
sistency of the cycle10 and age of onset of menstrual
cycle70), genetic factors such as hypermobility,6,16,36,67 caloric
intake,5,52 and activity levels53 are all factors that may
influence a subjects knee laxity response but are difficult
to control.
Limitations of the Study
Despite its limitations, the current study found a significant
influence of hormones on the structural and mechanical
properties of the knee in a majority of the subjects studied.
One assumption in this study was that subjects were
relaxed during laxity testing. Activating muscles during the
laxity testing would increase resistance to the loading,
which in turn would decrease laxity and increase stiffness.
Measuring quadriceps and hamstring muscle activity with
an arthrometer would confirm this assumption and provide
more reliable results.34 A second variable that may influ-
ence knee joint laxity, especially in the ACL, is water con-
tent. Approximately two thirds of the ACL wet weight is
water, which may make a marked contribution to its nonlin-
ear viscoelastic behavior.13 It is possible that an increased
body weight at the time of testing may reflect an increase in
water content and thus an increased laxity of the knee.
Consequently, a measure of total body water,29 to account for
this variable, may be a valuable addition to future studies.
An additional limitation of the current study is related to
the speed of loading during laxity testing, as all viscoelastic
materials demonstrate rate-dependent behavior.72 Thus, all
the knee joint measurements in this study were performed
by 1 experimenter to minimize loading rate inconsistencies.
It was impossible to randomize the order of the 3 tests in
knee laxity, as the data were collected during 1 menstrual
cycle. Randomizing the data collection across more than 1
menstrual cycle could remove any bias caused by either a
subjects learning-effect response or the examiners knowl-
edge of the subjects menstrual phase.
Furthermore, we recruited participants who were
actively involved in sporting activity at a recreational
level, but there are differences between recreational level
athletes and top athletes in terms of both knee laxity45 and
movement patterns.64 Because studies found that the inci-
dence of ACL injury was highest in female athletes playing
at the top level, testing top athletes may be more appropri-
ate for understanding of the ACL injury mechanism during
sports activity.56 However, it was difficult to find a large
number of top athletes who met our subject criteria, such
as no previous knee injury and no oral contraceptive use.
Also, multiple data collections at specific times during the
menstrual cycles of the subjects made recruitment of high
level athletes during their athletic season difficult.
A limitation of the KT-2000 arthrometer is that it requires
compression of soft tissue, leading to possible overestimation
of the displacement load.34 This error may be related to sub-
ject size as well as body composition.
Clinical Relevance: Effects of Knee Joint
Laxity and Stiffness on Knee Joint Function
In the current study, large variations in knee laxity response
at 89 N were observed ranging from 0.14 to 3.79 mm.
However, with a side-to-side difference of more than 3 mm of
the anterior tibial translation indicating ACL rupture,14,66 it
is possible that a difference in knee laxity of 3 mm during the
menstrual cycle may result in decreased functionality of the
knee, indicating a higher risk of ACL injury. Studies have
speculated that functional outcomes after ACL injury may
be correlated to passive knee joint laxity.24,66 In addition, pas-
sive knee laxity measurement has been used as a diagnostic
tool to evaluate successful knee joint function after rehabili-
tation with or without surgery.66 Wojtys and Huston75 have
indicated that muscle timing and recruitment patterns in
response to anterior tibial translation are altered after ACL
injury. The timing of muscle response to anterior tibial
596 Park et al The American Journal of Sports Medicine
t ranslation was delayed in ACL-injured patients, and muscle
timing and recruitment pattern are more likely to be affected
by a patients activity and skill levels.75 Similar to the rela-
tionship between knee joint laxity and functional outcomes
after ACL injury, several studies have also observed decreased
knee joint function with an increased passive knee joint
laxity.31,59 Female subjects with greater knee laxity demon-
strate greater electromyography (EMG) peak amplitude
during landing from a jump, a longer time to detect joint
motion in proprioception tests,59 and delayed generation of
muscle torque in isokinetic dynamometer testing compared
to their male counterparts.31 Thus, there is speculation that
a decreased protective mechanism caused by the increased
knee laxity of females may increase ACL injury risk during
physical activity.61 However, contributions of variations in
passive knee joint laxity during the menstrual cycle to
dynamic knee joint function have not been investigated.
It has been suggested that knee joint stiffness can be an
important diagnostic variable for assessing ligament
injury.40,42 Markolf et al42 found that ACL-injured knees have
increased passive knee laxity and decreased stiffness com-
pared with uninjured knees in 35 patients. They found that
changes in knee stiffness were most apparent at low loads of
100 N or less.42 Furthermore, during sports, ACL injury
coincides with peak ACL strains at 40 to 70 ms after external
loading is applied at the knee.80 Thus, changes in knee stiff-
ness may be an important factor related to ACL injury risk
in dynamic situations because the knee structure must
adapt very quickly, in a fraction of a second.59
A 50% to 77% reduction in knee stiffness in ACL-injured
subjects has been reported.40,42 However, commonly available
stiffness measurements, given only at specific loads (45, 89,
and 133 N), are not sensitive enough to detect small changes
in the material properties of knee structures.35 It has been
reported that one half or less of ACL ruptures showed similar
force-displacement behavior compared with a normal knee.35
The current technique using a quadratic function to calculate
instantaneous stiffness (first derivative) and the change in
stiffness (second derivative) enables differentiation of knee
stiffness decreases in the midportion of the force-displace-
ment curve. A reduction in knee stiffness of approximately
17% was seen during ovulation compared with the luteal
phase. There is a similar reduction of stiffness at the terminal
portion of the force-displacement curve in a tear of at most
25% of the ACL.35 Furthermore, subject variations in the
turning points (the peak of the second derivative: minima
and maxima points) indicate transitions in the ligamentous
force-strain characteristics. As the current stiffness measure-
ment was able to capture critical low load behavior of the
force-displacement curve, this novel method may be clinically
effective in predicting dynamic functionality of the joint and
diagnosis of partial ACL injury at low loads. However,
whether changes in stiffness affect dynamic joint function
has not been investigated in terms of ACL injury risk.
CONCLUSION
We report that hormone levels are related to increased
knee laxity during ovulation and identify an antagonistic
influence of estradiol and progesterone on knee laxity and
stiffness during the luteal phase. We also suggest that
changes in knee stiffness demonstrated with a new compu-
tational approach provide important information on the
load behavior of the knee structure.
This study did not address the question of whether hor-
mone influences on knee laxity also result in changes in
lower extremity biomechanics, such as movement or mus-
cular controls during the menstrual cycle. To date, most
studies have focused on the effects of changing hormones
on joint laxity or the influence of joint laxity on the fre-
quency of injury in female athletes. Yet there have been
few studies on the effects of hormones on the biomechani-
cal and neuromuscular changes in female athletes during
the menstrual cycle.1,12,27 No studies have investigated
whether changes in knee joint laxity during the menstrual
cycle affect lower extremity mechanics in sporting activi-
ties. Therefore, the way in which these biomechanical and
neuromuscular changes associated with knee joint laxity
contribute to a higher injury incident rate in female
athletes is unknown. The next step in the continuation of
this study will be to quantify changes in lower extremity
biomechanics corresponding to changes in knee laxity at
similar time points in the menstrual cycle.
ACKNOWLEDGMENT
This work was supported by the Institute for Gender
and Health (IGH) of the Canadian Institutes of Health and
Research (CIHR). The authors thank Erica Enevold and
Mandy Chan for blood draw, Dr Tak Fung for his support
in statistical analysis, as well as Colin Roeke for his cre-
ation of the figures.
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APPENDIX
Comparisons of R2 Value Between a New and Typical Stiffness Calculationa

Stiffness 1 Stiffness 2 Stiffness 3

New calculation .976 (.030) .984 (.009) .975 (.020)

Typical calculation .961 (.045) .968 (.031) .973 (.017)
a
Data are mean (SD). New calculation: the local minima and maxima points of the second derivative of the load-displacement curve were
determined for stiffness calculations. Stiffness 1, linear stiffness between the origin and the local minima point of the curve; stiffness 2,
linear stiffness between the local minima and maxima points of the curve; stiffness 3, linear stiffness between the maxima point and the
end point of the curve. Typical calculation: 45 N and 89 N of the load-displacement curve were used as reference points to calculate regional
linear stiffness. Stiffness 1, linear stiffness between the origin and 45 N of the curve; stiffness 2, linear stiffness between 45 N and 89 N of
the curve; stiffness 3, linear stiffness between 89 N and the end point of the curve.