# ...
open my $in1, "<", $file1 or die "unable to open $file1 $!";
open my $in2, "<", $file2 or die "unable to open $file2 $!";
open my $out, '>', $file3 or die "unable to open $file3 $!";

my $header1 = <$in1>;
my $header2 = <$in2>;
print $out "some header for output file \n";
# now you are all set with headers, you can focus on the data

while (my $line1 = <in1>) {
     my $line2 = <in2>;
     last unless defined $line2;
     chomp $line1, $line2;
     my @values1 = split $line1;
     my @values2 = split $line2;
     my @values3 = map {$values1[$_] - $values2[$_]} 0..$#values1;
     print $out "@values3 \n";
}