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Three ways to do a two-way ANOVA with Python

In an earlier post I showed four different techniques that enables one-way analysis of variance (ANOVA) using Python.  In this post we are going to learn how to do two-way ANOVA for independent measures using Python.

First, we ar going to learn how to calculate the ANOVA table “by hand”. Second, we are going to use Statsmodels and, third, we carry out the ANOVA in Python using pyvttbl. Finally, as a bonus, we will also use Pingouin Stats, a newer Python package.

An important advantage of the two-way ANOVA is that it is more efficient compared to the one-way. There are two assignable sources of variation – supp and dose in our example – and this helps to reduce error variation thereby making this design more efficient.

Two-way ANOVA (factorial) can be used to, for instance, compare the means of populations that are different in two ways. It can also be used to analyse the mean responses in an experiment with two factors. Unlike One-Way ANOVA, it enables us to test the effect of two factors at the same time.

One can also test for independence of the factors provided there are more than one observation in each cell. The only restriction is that the number of observations in each cell has to be equal (there is no such restriction in case of one-way ANOVA).

We discussed linear models earlier – and ANOVA is indeed a kind of linear model – the difference being that ANOVA is where you have discrete factors whose effect on a continuous (variable) result you want to understand.

Python 2-way ANOVA

import pandas as pd
import statsmodels.api as sm from statsmodels.formula.api import ols from statsmodels.stats.anova import anova_lm from import interaction_plot import matplotlib.pyplot as plt from scipy import stats

In the code above we import all the needed Python libraries and methods for doing the two first methods using Python (calculation with Python and using Statsmodels). In the last, and third, method for doing python ANOVA we are going to use Pyvttbl. As in the previous post on one-way ANOVA using Python we will use a set of data that is available in R but can be downloaded here: TootGrowth Data. Pandas is used to create a dataframe that is easy to manipulate.

datafile = "ToothGrowth.csv"
data = pd.read_csv(datafile)

It can be good to explore data before continuing with the inferential statistics. statsmodels has methods for visualising factorial data. We are going to use the method interaction_plot.

fig = interaction_plot(data.dose, data.supp, data.len,
             colors=['red','blue'], markers=['D','^'], ms=10)
Python ANOVA Interaction Plot
Interaction plot using Statsmodels

Calculation of Sum of Squares

The calculations of the sum of squares (the variance in the data) is quite simple using Python. First we start with getting the sample size (N) and the degree of freedoms needed. We will use them later to calculate the mean square. After we have the degree of freedom we continue with calculation of the sum of squares.

Degrees of Freedom

N = len(data.len)
df_a = len(data.supp.unique()) - 1
df_b = len(data.dose.unique()) - 1
df_axb = df_a*df_b 
df_w = N - (len(data.supp.unique())*len(data.dose.unique()))

Sum of Squares

Python ANOVA - partitioning of the Sum of Squares for a 2-way ANOVA
partitioning of the Sum of Squares (SS)

For the calculation of the sum of squares A, B and Total we will need to have the grand mean. Using Pandas DataFrame method mean on the dependent variable only will give us the grand mean:

grand_mean = data['len'].mean()

The grand mean is simply the mean of all scores of len.

Sum of Squares A – supp

We start with calculation of Sum of Squares for the factor A (supp).

ssq_a = sum([(data[data.supp ==l].len.mean()-grand_mean)**2 for l in data.supp])
Sum of Squares B – dose

Calculation of the second Sum of Square, B (dose), is pretty much the same but over the levels of that factor.

ssq_b = sum([(data[data.dose ==l].len.mean()-grand_mean)**2 for l in data.dose])
Sum of Squares Total
ssq_t = sum((data.len - grand_mean)**2)
Sum of Squares Within (error/residual)

Next, we need to calculate the Sum of Squares Within which is sometimes referred to as error or residual.

vc = data[data.supp == 'VC']
oj = data[data.supp == 'OJ']
vc_dose_means = [vc[vc.dose == d].len.mean() for d in vc.dose]
oj_dose_means = [oj[oj.dose == d].len.mean() for d in oj.dose]
ssq_w = sum((oj.len - oj_dose_means)**2) +sum((vc.len - vc_dose_means)**2)
Sum of Squares interaction

Since we have a two-way design we need to calculate the Sum of Squares for the interaction of A and B.

ssq_axb = ssq_t-ssq_a-ssq_b-ssq_w

Mean Squares

We continue with the calculation of the mean square for each factor, the interaction of the factors, and within.

Mean Square A
ms_a = ssq_a/df_a
Mean Square B
ms_b = ssq_b/df_b
Mean Square AxB
ms_axb = ssq_axb/df_axb
Mean Square Within/Error/Residual
ms_w = ssq_w/df_w


The F-statistic is simply the mean square for each effect and the interaction divided by the mean square for within (error/residual).

f_a = ms_a/ms_w
f_b = ms_b/ms_w
f_axb = ms_axb/ms_w

Obtaining p-values

We can use the scipy.stats method f.sf to check if our obtained F-ratios is above the critical value. Doing that we need to use our F-value for each effect and interaction as well as the degrees of freedom for them, and the degree of freedom within.

p_a = stats.f.sf(f_a, df_a, df_w)
p_b = stats.f.sf(f_b, df_b, df_w)
p_axb = stats.f.sf(f_axb, df_axb, df_w)

The results are, right now, stored in a lot of variables. To obtain a  morereadable result we can create a DataFrame that will contain our ANOVA table.

results = {'sum_sq':[ssq_a, ssq_b, ssq_axb, ssq_w],
           'df':[df_a, df_b, df_axb, df_w],
           'F':[f_a, f_b, f_axb, 'NaN'],
            'PR(>F)':[p_a, p_b, p_axb, 'NaN']}
columns=['sum_sq', 'df', 'F', 'PR(>F)']

aov_table1 = pd.DataFrame(results, columns=columns,
                          index=['supp', 'dose', 
                          'supp:dose', 'Residual'])

As a Psychologist most of the journals we publish in requires to report effect sizes. Common software, such as, SPSS have eta squared as output. However, eta squared is an overestimation of the effect. To get a less biased effect size measure we can use omega squared. The following two functions adds eta squared and omega squared to the above DataFrame that contains the  ANOVA table.

def eta_squared(aov):
    aov['eta_sq'] = 'NaN'
    aov['eta_sq'] = aov[:-1]['sum_sq']/sum(aov['sum_sq'])
    return aov

def omega_squared(aov):
    mse = aov['sum_sq'][-1]/aov['df'][-1]
    aov['omega_sq'] = 'NaN'
    aov['omega_sq'] = (aov[:-1]['sum_sq']-(aov[:-1]['df']*mse))/(sum(aov['sum_sq'])+mse)
    return aov


Output ANOVA table

Two-way ANOVA using Statsmodels

There is, of course, a much easier way to do Two-way ANOVA with Python. We can use Statsmodels which have a similar model notation as many R-packages (e.g., lm). We start with formulation of the model:

formula = 'len ~ C(supp) + C(dose) + C(supp):C(dose)'
model = ols(formula, data).fit()
aov_table = anova_lm(model, typ=2)

Statsmodels does not calculate effect sizes for us. My functions above can, again, be used and will add omega and eta squared effect sizes to the ANOVA table. Actually, I created these two functions to enable calculation of omega and eta squared effect sizes on the output of Statsmodels anova_lm method.


Output ANOVA table

What is neat with using statsmodels is that we can also do some diagnostics. It is, for instance, very easy to take our model fit (the linear model fitted with the OLS method) and get a Quantile-Quantile (QQplot):

res = model.resid 
fig = sm.qqplot(res, line='s')
Python ANOVA QQplot Statsmodels
QQplot using Statsmodels

Two-way ANOVA in Python using pyvttbl

The third way to do Python ANOVA is using the library pyvttbl.  Pyvttbl has its own method (also called DataFrame) to create data frames.

from pyvttbl import DataFrame
df['id'] = xrange(len(df['len']))

print(df.anova('len', sub='id', bfactors=['supp', 'dose']))

The ANOVA tables of Pyvttbl contains a lot of more information compared to that of statsmodels. Actually, Pyvttbl output contains an effect size measure; the generalized omega squared.

Measure: len

Source Type III Sum of Squares df MS F Sig. η2G Obs. SE of x̄ ±95% CI λ Obs. Power
supp 205.350 1.000 205.350 15.572 0.000 0.224 30.000 0.678 1.329 8.651 0.823
dose 2426.434 2.000 1213.217 92.000 0.000 0.773 20.000 0.831 1.628 68.148 1.000
supp * dose 108.319 2.000 54.159 4.107 0.022 0.132 10.000 1.175 2.302 1.521 0.173
Error 712.106 54.000 13.187                
Total 3452.209 59.000                  


Two-Way ANOVA using Pingouin (Bonus)

Here’s a bonus method to carry out ANOVA using Python; using the Python package Pingouin. Although pyvttbl is quite good, it’s not maintained any more. Here, Pingouin offers a very easy way to for ANOVA in Python.

import pandas as pd
import pingouin as pg

data = ''

df = pd.read_csv(data, index_col=0)
aov = pg.anova(dv='len', between=['supp', 'dose'], data=df,



    • Hey André! I am glad you found it useful! You’re right! Thanks for correcting my mistake. It is now changed. Have a nice day!

  1. Alina Alina

    Great post! This was the first one I’ve found that clearly and succinctly explained exactly what I’m trying to do–many thanks!

  2. Xiayu Chen Xiayu Chen

    Hi, Erik, Thanks a lot for your post! It definitely helps me solve two-way anova with python programming.
    Additionally, I guess that you have omitted ‘f_axb’ at the step of F-ratio calculation?

  3. Abrams Barber Abrams Barber

    Thank you, this was useful, but definition of f_axb seems be lost.
    Can you add expression?

    • Hey,

      Thanks for spotting this. I’ve added the calculation now.

  4. Abhishek Abhishek

    Can the two way annova be calculated using the spicy package????? Can you please help me with that.

    • A spicy package would be cool! See my reply to your corretion.

  5. Abhishek Abhishek

    Sorry i meant scipy package??

    • Hey. As far as I am aware, there’s no 2-way ANOVA method (e.g., scipy.stats.f_oneway) in the SciPy package. You could, of course, do the calculations using SciPy.

  6. Lilta Alem Lilta Alem

    Dear Erik Marsja, PhD,
    In the beginning of this month, I sent you message seeking your assistance to resolve errors I encountered when I tried to Two Way ANOVA analysis. Still, I need help in this regard. If possible I want you to arrange two or three tutorial sessions online, I can pay for the service. I’m older men a new for Python, but familiar with statistics, I want to return to work after equipping with Python training. I look forward to hearing from you.

    With regards,

    • That is a good suggestion. I’ve added how to do this in another post and will update this post today, also, with a code snippet.

      Thanks, Mikael,



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