Lab Overview

Time: ~30 minutes

Goal: Practice one-way ANOVA analysis from start to finish using real public health data

Learning Objectives:

  • Understand when and why to use ANOVA instead of multiple t-tests
  • Set up hypotheses for ANOVA
  • Conduct and interpret the F-test
  • Perform post-hoc tests when appropriate
  • Check ANOVA assumptions
  • Calculate and interpret effect size (η²)

Structure:

  • Part A: Guided Example (follow along)
  • Part B: Your Turn (independent practice)

Submission: Upload your completed .Rmd file and published to Brightspace by the end of class.


PART A: GUIDED EXAMPLE

Example: Blood Pressure and BMI Categories

Research Question: Is there a difference in mean systolic blood pressure (SBP) across three BMI categories (Normal weight, Overweight, Obese)?

Why ANOVA? We have one continuous outcome (SBP) and one categorical predictor with THREE groups (BMI category). Using multiple t-tests would inflate our Type I error rate.


Step 1: Setup and Data Preparation

# Load necessary libraries
library(tidyverse)   # For data manipulation and visualization
library(knitr)       # For nice tables
library(car)         # For Levene's test
library(NHANES)      # NHANES dataset

# Load the NHANES data
data(NHANES)

Create analysis dataset:

# Set seed for reproducibility
set.seed(553)

# Create BMI categories and prepare data
bp_bmi_data <- NHANES %>%
  filter(Age >= 18 & Age <= 65) %>%  # Adults 18-65
  filter(!is.na(BPSysAve) & !is.na(BMI)) %>%
  mutate(
    bmi_category = case_when(
      BMI < 25 ~ "Normal",
      BMI >= 25 & BMI < 30 ~ "Overweight",
      BMI >= 30 ~ "Obese",
      TRUE ~ NA_character_
    ),
    bmi_category = factor(bmi_category, 
                         levels = c("Normal", "Overweight", "Obese"))
  ) %>%
  filter(!is.na(bmi_category)) %>%
  select(ID, Age, Gender, BPSysAve, BMI, bmi_category)

# Display first few rows
head(bp_bmi_data) %>% 
  kable(caption = "Blood Pressure and BMI Dataset (first 6 rows)")
Blood Pressure and BMI Dataset (first 6 rows)
ID Age Gender BPSysAve BMI bmi_category
51624 34 male 113 32.22 Obese
51624 34 male 113 32.22 Obese
51624 34 male 113 32.22 Obese
51630 49 female 112 30.57 Obese
51647 45 female 118 27.24 Overweight
51647 45 female 118 27.24 Overweight
# Check sample sizes
table(bp_bmi_data$bmi_category)
## 
##     Normal Overweight      Obese 
##       1939       1937       2150

Interpretation: We have 6026 adults with complete BP and BMI data across three BMI categories.


Step 2: Descriptive Statistics

# Calculate summary statistics by BMI category
summary_stats <- bp_bmi_data %>%
  group_by(bmi_category) %>%
  summarise(
    n = n(),
    Mean = mean(BPSysAve),
    SD = sd(BPSysAve),
    Median = median(BPSysAve),
    Min = min(BPSysAve),
    Max = max(BPSysAve)
  )

summary_stats %>% 
  kable(digits = 2, 
        caption = "Descriptive Statistics: Systolic BP by BMI Category")
Descriptive Statistics: Systolic BP by BMI Category
bmi_category n Mean SD Median Min Max
Normal 1939 114.23 15.01 113 78 221
Overweight 1937 118.74 13.86 117 83 186
Obese 2150 121.62 15.27 120 82 226

Observation: The mean SBP appears to increase from Normal (114.2) to Overweight (118.7) to Obese (121.6).

But is this difference statistically significant?


Step 3: Visualize the Data

# Create boxplots with individual points
ggplot(bp_bmi_data, 
  aes(x = bmi_category, y = BPSysAve, fill = bmi_category)) +
  geom_boxplot(alpha = 0.7, outlier.shape = NA) +
  geom_jitter(width = 0.2, alpha = 0.1, size = 0.5) +
  scale_fill_brewer(palette = "Set2") +
  labs(
    title = "Systolic Blood Pressure by BMI Category",
    subtitle = "NHANES Data, Adults aged 18-65",
    x = "BMI Category",
    y = "Systolic Blood Pressure (mmHg)",
    fill = "BMI Category"
  ) +
  theme_minimal(base_size = 12) +
  theme(legend.position = "none")

What the plot tells us:

  • There appears to be a trend: higher BMI categories have higher median SBP
  • The boxes overlap, but the obese group appears shifted upward
  • Variability (box heights) looks similar across groups

Step 4: Set Up Hypotheses

Null Hypothesis (H₀): μ_Normal = μ_Overweight = μ_Obese
(All three population means are equal)

Alternative Hypothesis (H₁): At least one population mean differs from the others

Significance level: α = 0.05


Step 5: Fit the ANOVA Model

# Fit the one-way ANOVA model
anova_model <- aov(BPSysAve ~ bmi_category, data = bp_bmi_data)

# Display the ANOVA table
summary(anova_model)
##                Df  Sum Sq Mean Sq F value Pr(>F)    
## bmi_category    2   56212   28106   129.2 <2e-16 ***
## Residuals    6023 1309859     217                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Interpretation:

  • F-statistic: 129.24
  • Degrees of freedom: df₁ = 2 (k-1 groups), df₂ = 6023 (n-k)
  • p-value: < 2e-16 (very small)
  • Decision: Since p < 0.05, we reject H₀
  • Conclusion: There is statistically significant evidence that mean systolic BP differs across at least two BMI categories.

Step 6: Post-Hoc Tests (Tukey HSD)

Why do we need this? The F-test tells us that groups differ, but not which groups differ. Tukey’s Honest Significant Difference controls the family-wise error rate for multiple pairwise comparisons.

# Conduct Tukey HSD test
tukey_results <- TukeyHSD(anova_model)
print(tukey_results)
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = BPSysAve ~ bmi_category, data = bp_bmi_data)
## 
## $bmi_category
##                       diff      lwr      upr p adj
## Overweight-Normal 4.507724 3.397134 5.618314     0
## Obese-Normal      7.391744 6.309024 8.474464     0
## Obese-Overweight  2.884019 1.801006 3.967033     0
# Visualize the confidence intervals
plot(tukey_results, las = 0)

Interpretation:

Comparison Mean Diff 95% CI p-value Significant?
Overweight - Normal 4.51 [3.4, 5.62] 1.98e-13 Yes
Obese - Normal 7.39 [6.31, 8.47] < 0.001 Yes
Obese - Overweight 2.88 [1.8, 3.97] 1.38e-09 Yes

Conclusion: All three pairwise comparisons are statistically significant. Obese adults have higher SBP than overweight adults, who in turn have higher SBP than normal-weight adults.


Step 7: Calculate Effect Size

# Extract sum of squares from ANOVA table
anova_summary <- summary(anova_model)[[1]]

ss_treatment <- anova_summary$`Sum Sq`[1]
ss_total <- sum(anova_summary$`Sum Sq`)

# Calculate eta-squared
eta_squared <- ss_treatment / ss_total

cat("Eta-squared (η²):", round(eta_squared, 4), "\n")
## Eta-squared (η²): 0.0411
cat("Percentage of variance explained:", round(eta_squared * 100, 2), "%")
## Percentage of variance explained: 4.11 %

Interpretation: BMI category explains 4.11% of the variance in systolic BP.

  • Effect size guidelines: Small (0.01), Medium (0.06), Large (0.14)
  • Our effect: Small

While statistically significant, the practical effect is modest—BMI category alone doesn’t explain most of the variation in blood pressure.


Step 8: Check Assumptions

ANOVA Assumptions:

  1. Independence: Observations are independent (assumed based on study design)
  2. Normality: Residuals are approximately normally distributed
  3. Homogeneity of variance: Equal variances across groups
# Create diagnostic plots
par(mfrow = c(2, 2))
plot(anova_model)

par(mfrow = c(1, 1))

Diagnostic Plot Interpretation:

  1. Residuals vs Fitted: Points show random scatter around zero with no clear pattern → Good!
  2. Q-Q Plot: Points follow the diagonal line reasonably well → Normality assumption is reasonable
  3. Scale-Location: Red line is relatively flat → Equal variance assumption is reasonable
  4. Residuals vs Leverage: No points beyond Cook’s distance lines → No highly influential outliers
# Levene's test for homogeneity of variance
levene_test <- leveneTest(BPSysAve ~ bmi_category, data = bp_bmi_data)
print(levene_test)
## Levene's Test for Homogeneity of Variance (center = median)
##         Df F value  Pr(>F)  
## group    2  2.7615 0.06328 .
##       6023                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Levene’s Test Interpretation:

  • p-value: 0.0633
  • If p < 0.05, we would reject equal variances
  • Here: Equal variance assumption is met

Overall Assessment: With n > 2000, ANOVA is robust to minor violations. Our assumptions are reasonably satisfied.


Step 9: Report Results

Example Results Section:

We conducted a one-way ANOVA to examine whether mean systolic blood pressure (SBP) differs across BMI categories (Normal, Overweight, Obese) among 6,026 adults aged 18-65 from NHANES. Descriptive statistics showed mean SBP of 114.2 mmHg (SD = 15) for normal weight, 118.7 mmHg (SD = 13.9) for overweight, and 121.6 mmHg (SD = 15.3) for obese individuals.

The ANOVA revealed a statistically significant difference in mean SBP across BMI categories, F(2, 6023) = 129.24, p < 0.001. Tukey’s HSD post-hoc tests indicated that all pairwise comparisons were significant (p < 0.05): obese adults had on average 7.4 mmHg higher SBP than normal-weight adults, and 2.9 mmHg higher than overweight adults.

The effect size (η² = 0.041) indicates that BMI category explains 4.1% of the variance in systolic blood pressure, representing a small practical effect. These findings support the well-established relationship between higher BMI and elevated blood pressure, though other factors account for most of the variation in SBP.


PART B: YOUR TURN - INDEPENDENT PRACTICE

Practice Problem: Physical Activity and Depression

Research Question: Is there a difference in the number of days with poor mental health across three physical activity levels (None, Moderate, Vigorous)?

Your Task: Complete the same 9-step analysis workflow you just practiced, but now on a different outcome and predictor.


Step 1: Data Preparation

# Prepare the dataset
set.seed(553)

mental_health_data <- NHANES %>%
  filter(Age >= 18) %>%
  filter(!is.na(DaysMentHlthBad) & !is.na(PhysActive)) %>%
  mutate(
    activity_level = case_when(
      PhysActive == "No" ~ "None",
      PhysActive == "Yes" & !is.na(PhysActiveDays) & PhysActiveDays < 3 ~ "Moderate",
      PhysActive == "Yes" & !is.na(PhysActiveDays) & PhysActiveDays >= 3 ~ "Vigorous",
      TRUE ~ NA_character_
    ),
    activity_level = factor(activity_level, 
                           levels = c("None", "Moderate", "Vigorous"))
  ) %>%
  filter(!is.na(activity_level)) %>%
  select(ID, Age, Gender, DaysMentHlthBad, PhysActive, activity_level)

# YOUR TURN: Display the first 6 rows and check sample sizes

# Display first few rows
head(mental_health_data) %>% 
  kable(caption = "Mental Health and Physical Activity Dataset (first 6 rows)")
Mental Health and Physical Activity Dataset (first 6 rows)
ID Age Gender DaysMentHlthBad PhysActive activity_level
51624 34 male 15 No None
51624 34 male 15 No None
51624 34 male 15 No None
51630 49 female 10 No None
51647 45 female 3 Yes Vigorous
51647 45 female 3 Yes Vigorous
# Check sample sizes
table(mental_health_data$activity_level)
## 
##     None Moderate Vigorous 
##     3139      768     1850

YOUR TURN - Answer these questions:

  • How many people are in each physical activity group?
    • None: 3139
    • Moderate: 768
    • Vigorous: 1850

Step 2: Descriptive Statistics

# YOUR TURN: Calculate summary statistics by activity level
# Hint: Follow the same structure as the guided example
# Variables to summarize: n, Mean, SD, Median, Min, Max
# Calculate summary statistics by activity level. 
summary_stats <- mental_health_data %>%
  group_by(activity_level) %>%
  summarise(
    n = n(),
    Mean = mean(DaysMentHlthBad),
    SD = sd(DaysMentHlthBad),
    Median = median(DaysMentHlthBad),
    Min = min(DaysMentHlthBad),
    Max = max(DaysMentHlthBad)
  )

summary_stats %>% 
  kable(digits = 2, 
        caption = "Descriptive Statistics: Poor Mental Health by activity level")
Descriptive Statistics: Poor Mental Health by activity level
activity_level n Mean SD Median Min Max
None 3139 5.08 9.01 0 0 30
Moderate 768 3.81 6.87 0 0 30
Vigorous 1850 3.54 7.17 0 0 30

YOUR TURN - Interpret:

  • Which group has the highest mean number of bad mental health days?
The group with the highest mean number of bad mental health days was those who had no physical activity.
  • Which group has the lowest?
The group with the lowest mean number of bad mental health days was those who completed vigorous exercise.

Step 3: Visualization

# YOUR TURN: Create boxplots comparing DaysMentHlthBad across activity levels
# Hint: Use the same ggplot code structure as the example
# Change variable names and labels appropriately
# Create boxplots with individual points
ggplot(mental_health_data, 
  aes(x = activity_level, y = DaysMentHlthBad, fill = activity_level)) +
  geom_boxplot(alpha = 0.7, outlier.shape = NA) +
  geom_jitter(width = 0.2, alpha = 0.1, size = 0.5) +
  scale_fill_brewer(palette = "Set2") +
  labs(
    title = "Days with Poor Mental Health by Physical Activity Level",
    subtitle = "NHANES Data, Adults aged 18-65",
    x = "Activity Level",
    y = "Poor Mental Health (Days)",
    fill = "Activity Level"
  ) +
  theme_minimal(base_size = 12) +
  theme(legend.position = "none")

YOUR TURN - Describe what you see:

  • Do the groups appear to differ?
At first glance, No physical activity and moderate activity appear to have a similar shape while vigorous activity has a much smaller box plot than the other two. While the first two boxes look the same, the first boxplot for no and vigorous physical activity has a lot more values spread out across the y axis than moderate physical activity. 
  • Are the variances similar across groups?
The variances of no and moderate physical activity have a very similar IQR and range while vigorous physical activity has a lower variance than the first two boxplots. 

Step 4: Set Up Hypotheses

YOUR TURN - Write the hypotheses:

**Null Hypothesis (H₀): μ_None = μ_Moderate = μ_Vigorous (All three population means are equal)

**Alternative Hypothesis (H₁): At least one population mean is different from the others

Significance level: α = 0.05


Step 5: Fit the ANOVA Model

# YOUR TURN: Fit the ANOVA model
# Outcome: DaysMentHlthBad
# Predictor: activity_level
# Fit the one-way ANOVA model
anova_model <- aov(DaysMentHlthBad ~ activity_level, data = mental_health_data)

# Display the ANOVA table
summary(anova_model)
##                  Df Sum Sq Mean Sq F value   Pr(>F)    
## activity_level    2   3109  1554.6   23.17 9.52e-11 ***
## Residuals      5754 386089    67.1                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

YOUR TURN - Extract and interpret the results:

  • F-statistic: 23.17
  • Degrees of freedom: 2
  • p-value: 9.52e-11 (very small)
  • Decision (reject or fail to reject H₀): reject the null hypothesis
  • Statistical conclusion in words: There is statistically significant evidence that mean days of poor mental health differs across at least two physical activity levels.

Step 6: Post-Hoc Tests

# YOUR TURN: Conduct Tukey HSD test
# Only if your ANOVA p-value < 0.05
tukey_results <- TukeyHSD(anova_model)
print(tukey_results)
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = DaysMentHlthBad ~ activity_level, data = mental_health_data)
## 
## $activity_level
##                         diff       lwr        upr     p adj
## Moderate-None     -1.2725867 -2.045657 -0.4995169 0.0003386
## Vigorous-None     -1.5464873 -2.109345 -0.9836298 0.0000000
## Vigorous-Moderate -0.2739006 -1.098213  0.5504114 0.7159887
# Visualize the confidence intervals
plot(tukey_results, las = 0)

YOUR TURN - Complete the table:

Comparison Mean Difference 95% CI Lower 95% CI Upper p-value Significant?
Moderate - None -1.27 -2.05 -0.50 0.0003 Yes
Vigorous - None -1.55 -2.11 -0.98 <0.0001 Yes
Vigorous - Moderate -0.27 -1.10 0.55 0.7160 No

Interpretation:

Which specific groups differ significantly?

Only two of the pairwise groups differed significantly. There is no statiscally siginifanct difference in mean poor mental health days between vigorous and moderate exercise. However, there is a statistically significant difference in mean poor mental health days between moderate and no activity, and vigorous activity. Individuals who completed moderate and vigorous exerise had less mean poor mental health days than those who completed no activity, but there was no significant decrease/increase in mean days with poor mental health depending on moderate or vigorous physical activity. 

Step 7: Calculate Effect Size

# YOUR TURN: Calculate eta-squared
# Hint: Extract Sum Sq from the ANOVA summary
# Extract sum of squares from ANOVA table
anova_summary <- summary(anova_model)[[1]]

ss_treatment <- anova_summary$`Sum Sq`[1]
ss_total <- sum(anova_summary$`Sum Sq`)

# Calculate eta-squared
eta_squared <- ss_treatment / ss_total

cat("Eta-squared (η²):", round(eta_squared, 4), "\n")
## Eta-squared (η²): 0.008
cat("Percentage of variance explained:", round(eta_squared * 100, 2), "%")
## Percentage of variance explained: 0.8 %

YOUR TURN - Interpret:

  • η² = 0.008
  • Percentage of variance explained: 0.8%
  • Effect size classification (small/medium/large): small
  • What does this mean practically?
Physical activity level explains 0.8% of the variance in mean days with poor mental health. The physical activity level alone does not explain most of the variation in poor mental health.

Step 8: Check Assumptions

# YOUR TURN: Create diagnostic plots
# Create diagnostic plots
par(mfrow = c(2, 2))
plot(anova_model)

par(mfrow = c(1, 1))

YOUR TURN - Evaluate each plot:

  1. Residuals vs Fitted:
Points show random scatter around zero with no clear pattern.
  1. Q-Q Plot:
Points do not fully follow the diagonal line and is right-skewed. However, sample size is very large (over 5,000), the Central Limit Theorem tells us ANOVA is robust to this violation.
  1. Scale-Location:
The red line is relatively flat → we can assume equal variance.
  1. Residuals vs Leverage:
There are no points beyond Cook's distance lines → No highly influential outliers.
# YOUR TURN: Conduct Levene's test
# Levene's test for homogeneity of variance
levene_test <- leveneTest(DaysMentHlthBad ~ activity_level, data = mental_health_data)
print(levene_test)
## Levene's Test for Homogeneity of Variance (center = median)
##         Df F value    Pr(>F)    
## group    2  23.168 9.517e-11 ***
##       5754                      
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

YOUR TURN - Overall assessment:

  • Are assumptions reasonably met?
With n > 2000, ANOVA is robust to minor violations of normality and differing results of Levene's test and Scale-Location plot.
  • Do any violations threaten your conclusions?
There is a violation with the Q-Q plot but since ANOVA is robust, we can still assume normality. There is a violation with the Levenes's Test that contradicts the Scale-Location plot. The p-value is 9.517e-11, which is lower than the significance level, so we would reject equal variances that was true in the Scale-Location plot. 

Step 9: Write Up Results

YOUR TURN - Write a complete 2-3 paragraph results section:

Include: 1. Sample description and descriptive statistics 2. F-test results 3. Post-hoc comparisons (if applicable) 4. Effect size interpretation 5. Public health significance

Your Results Section:

A one-way ANOVA test to determine whether mean days with poor (bad) mental health is different across physical activity level (None, Moderate, Vigorous) among 5,757 adults aged 18-65 from NHANES. Descriptive statistics showed a mean number of poor mental health days of 5.08 days (SD = 9.01) for no physical activity, 3.81 days (SD = 6.87) for moderate physcial activity, and 3.54 (SD = 7.17) days for vigorous physical exercise.

The ANOVA test showed a statistically significant difference in mean number of days with poor mental health across physical activity level, F(2,5754) = 23.17,p < 0.0001. The Tukey's HSD post-hoc tests showed that two of the three pairwise comparisons were significant (p<0.05): those who completed vigorous physical exercise had on average 1.55 less number of days with poor mental health compared to adults who completed no physical exercise and those who completed moderate physical exercise had an average of 1.27 less poor mental health days compared to those who completed no physical activity. There was not a significant difference in the pairwise comparison of vigorous to moderate activity. 

The effect size (η² = 0.008) shows that physical activity levels explain 0.8% of the variance in poor mental health days, meaning a small practical effect.  Increasing activity from none to moderate or vigorous leads to less days with poor mental health but not significantly when transitioning from moderate to vigorous activity. These findings support the relationship between physical activity and poor mental health, though other factors could be responsible for most of the variation in poor mental health days.

Reflection Questions

1. How does the effect size help you understand the practical vs. statistical significance?

It shows us that while our findings may be "statitically" significant, there may be other factors not measured that account for more of the reason why it is significant. We can use effect size to determine this and give practical context to our findings.

2. Why is it important to check ANOVA assumptions? What might happen if they’re violated?

It is important to check ANOVA assumptions to make sure our test was correct. If they were violated, then the ANOVA test may give the wrong conclusions. 

3. In public health practice, when might you choose to use ANOVA?

In public health practice, ANOVA might be used to compare group means to determine if at least one is different. For example, you may want to determine if mean birth weight differs across levels of maternal smoking (none, moderate, high), using ANOVA would be a way to determine if at least one of the levels differ. 

4. What was the most challenging part of this lab activity?

The most challenging part of this lab activity was checking the ANOVA assumptions. I had a hard time determing if they were correct or not. 

Submission Checklist

Before submitting, verify you have:

To submit: Upload both your .Rmd file and the HTML output to Brightspace.


Lab completed on: February 05, 2026


GRADING RUBRIC (For TA Use)

Total Points: 15

Category Criteria Points Notes
Code Execution All code chunks run without errors 4 - Deduct 1 pt per major error
- Deduct 0.5 pt per minor warning
Completion All “YOUR TURN” sections attempted 4 - Part B Steps 1-9 completed
- All fill-in-the-blank answered
- Tukey table filled in
Interpretation Correct statistical interpretation 4 - Hypotheses correctly stated (1 pt)
- ANOVA results interpreted (1 pt)
- Post-hoc results interpreted (1 pt)
- Assumptions evaluated (1 pt)
Results Section Professional, complete write-up 3 - Includes descriptive stats (1 pt)
- Reports F-test & post-hoc (1 pt)
- Effect size & significance (1 pt)

Detailed Grading Guidelines

Code Execution (4 points):

  • 4 pts: All code runs perfectly, produces correct output
  • 3 pts: Minor issues (1-2 small errors or warnings)
  • 2 pts: Several errors but demonstrates understanding
  • 1 pt: Major errors, incomplete code
  • 0 pts: Code does not run at all

Completion (4 points):

  • 4 pts: All sections attempted thoughtfully
  • 3 pts: 1-2 sections incomplete or minimal effort
  • 2 pts: Several sections missing
  • 1 pt: Only partial completion
  • 0 pts: Little to no work completed

Interpretation (4 points):

  • 4 pts: All interpretations correct and well-explained
  • 3 pts: Minor errors in interpretation
  • 2 pts: Several interpretation errors
  • 1 pt: Significant misunderstanding of concepts
  • 0 pts: No interpretation provided

Results Section (3 points):

  • 3 pts: Publication-quality, complete results section
  • 2 pts: Good but missing some elements
  • 1 pt: Incomplete or poorly written
  • 0 pts: No results section written

Common Deductions

  • -0.5 pts: Missing sample sizes in write-up
  • -0.5 pts: Not reporting confidence intervals
  • -1 pt: Incorrect hypothesis statements
  • -1 pt: Misinterpreting p-values
  • -1 pt: Not checking assumptions
  • -0.5 pts: Poor formatting (no tables, unclear output)