OK not shortest :-/ but fastest :-), vis:

print "Writing file...\n";
open FILE, ">test.txt" or die $!;
my $line = join "\t", (1..1000);
print FILE $line, "\n" for (1..600);
close FILE;
print "File written!\n";

# Original method
open PROFILES, "<test.txt" or die $!;
my @matrix = ();
my $start = time;
while (<PROFILES>) {
 @profile = split (/\t/, $_);
 while ($j <= $#profile) {
  $matrix[$j]->[$i] = $profile[$i];
  $j++;
 }
 $i++;
 $j = 0;
}
print "Original method takes ", time-$start, " seconds\n";
close PROFILES;

# my method
open PROFILES, "<test.txt" or die $!;
@matrix = ();
$start = time;
while(<PROFILES>){
    push @matrix, [split"\t"];
}
print "My method takes ", time-$start, " seconds\n";
close PROFILES;

# Blakem's method
open PROFILES, "<test.txt" or die $!;
@matrix = ();
$start = time;
@matrix = map[split],<PROFILES>;
print "Blakem's method takes ", time-$start, " seconds\n";
close PROFILES;

__DATA__
C:\>perl matrix.pl
Writing file...
File written!
Original method takes 18 seconds
My method takes 7 seconds
Blakem's method takes 52 seconds
[download]

PS I slightly modified the original code to remove the infinite loop so it actually will work to test it.

cheers

tachyon

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